Journal articles: 'Cargo receptor'

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Lopez, Sergio, Ana Maria Perez-Linero, Javier Manzano-Lopez, Susana Sabido-Bozo, Alejandro Cortes-Gomez, Sofia Rodriguez-Gallardo, Auxiliadora Aguilera-Romero, Veit Goder, and Manuel Muñiz. "Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum." Cells 9, no. 5 (May 22, 2020): 1295. http://dx.doi.org/10.3390/cells9051295.

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The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.

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Liu, Allen P., François Aguet, Gaudenz Danuser, and Sandra L. Schmid. "Local clustering of transferrin receptors promotes clathrin-coated pit initiation." Journal of Cell Biology 191, no. 7 (December 27, 2010): 1381–93. http://dx.doi.org/10.1083/jcb.201008117.

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Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

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Soohoo, Amanda L., and Manojkumar A. Puthenveedu. "Divergent modes for cargo-mediated control of clathrin-coated pit dynamics." Molecular Biology of the Cell 24, no. 11 (June 2013): 1725–34. http://dx.doi.org/10.1091/mbc.e12-07-0550.

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Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such “cargo-mediated regulation” of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the μ-opioid receptor, a physiologically relevant G protein–coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.

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Nie, Chao, Huimin Wang, Rui Wang, David Ginsburg, and Xiao-Wei Chen. "Dimeric sorting code for concentrative cargo selection by the COPII coat." Proceedings of the National Academy of Sciences 115, no. 14 (March 19, 2018): E3155—E3162. http://dx.doi.org/10.1073/pnas.1704639115.

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The flow of cargo vesicles along the secretory pathway requires concerted action among various regulators. The COPII complex, assembled by the activated SAR1 GTPases on the surface of the endoplasmic reticulum, orchestrates protein interactions to package cargos and generate transport vesicles en route to the Golgi. The dynamic nature of COPII, however, hinders analysis with conventional biochemical assays. Here we apply proximity-dependent biotinylation labeling to capture the dynamics of COPII transport in cells. When SAR1B was fused with a promiscuous biotin ligase, BirA*, the fusion protein SAR1B-BirA* biotinylates and thus enables the capture of COPII machinery and cargos in a GTP-dependent manner. Biochemical and pulse–chase imaging experiments demonstrate that the COPII coat undergoes a dynamic cycle of engagement–disengagement with the transmembrane cargo receptor LMAN1/ERGIC53. LMAN1 undergoes a process of concentrative sorting by the COPII coat, via a dimeric sorting code generated by oligomerization of the cargo receptor. Similar oligomerization events have been observed with other COPII sorting signals, suggesting that dimeric/multimeric sorting codes may serve as a general mechanism to generate selectivity of cargo sorting.

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Ren, Jihui, Younghoon Kee, Jon M. Huibregtse, and Robert C. Piper. "Hse1, a Component of the Yeast Hrs-STAM Ubiquitin-sorting Complex, Associates with Ubiquitin Peptidases and a Ligase to Control Sorting Efficiency into Multivesicular Bodies." Molecular Biology of the Cell 18, no. 1 (January 2007): 324–35. http://dx.doi.org/10.1091/mbc.e06-06-0557.

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Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.

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Ishikawa, Kuniko, Natalie L. Catlett, Jennifer L. Novak, Fusheng Tang, Johnathan J. Nau, and Lois S. Weisman. "Identification of an organelle-specific myosin V receptor." Journal of Cell Biology 160, no. 6 (March 17, 2003): 887–97. http://dx.doi.org/10.1083/jcb.200210139.

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Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p–Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.

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SZYMKIEWICZ, Iwona, Oleg SHUPLIAKOV, and Ivan DIKIC. "Cargo- and compartment-selective endocytic scaffold proteins." Biochemical Journal 383, no. 1 (September 24, 2004): 1–11. http://dx.doi.org/10.1042/bj20040913.

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The endocytosis of membrane receptors is a complex and tightly controlled process that is essential for maintaining cellular homoeostasis. The removal of receptors from the cell surface can be constitutive or ligand-induced, and occurs in a clathrin-dependent or -independent manner. The recruitment of receptors into specialized membrane domains, the formation of vesicles and the trafficking of receptors together with their ligands within endocytic compartments are regulated by reversible protein modifications, and multiple protein–protein and protein–lipid interactions. Recent reports describe a variety of multidomain molecules that facilitate receptor endocytosis and function as platforms for the assembly of protein complexes. These scaffold proteins typically act in a cargo-specific manner, recognizing one or more receptor types, or function at the level of endocytic cellular microcompartments by controlling the movement of cargo molecules and linking endocytic machineries to signalling pathways. In the present review we summarize present knowledge on endocytic scaffold molecules and discuss their functions.

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Nyfeler, Beat, Veronika Reiterer, Markus W. Wendeler, Eduard Stefan, Bin Zhang, Stephen W. Michnick, and Hans-Peter Hauri. "Identification of ERGIC-53 as an intracellular transport receptor of α1-antitrypsin." Journal of Cell Biology 180, no. 4 (February 18, 2008): 705–12. http://dx.doi.org/10.1083/jcb.200709100.

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Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

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Lam, Vinh Q., David Akopian, Michael Rome, Doug Henningsen, and Shu-ou Shan. "Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting." Journal of Cell Biology 190, no. 4 (August 23, 2010): 623–35. http://dx.doi.org/10.1083/jcb.201004129.

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The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting.

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Chen, Qingzhou, Junlin Teng, and Jianguo Chen. "ATL3, a cargo receptor for reticulophagy." Autophagy 15, no. 8 (April 28, 2019): 1465–66. http://dx.doi.org/10.1080/15548627.2019.1609862.

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11

Mitrovic, Sandra, Houchaima Ben-Tekaya, Eva Koegler, Jean Gruenberg, and Hans-Peter Hauri. "The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi." Molecular Biology of the Cell 19, no. 5 (May 2008): 1976–90. http://dx.doi.org/10.1091/mbc.e07-10-0989.

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Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.

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Kunduri, Govind, Changqing Yuan, Velayoudame Parthibane, Katherine M. Nyswaner, Ritu Kanwar, Kunio Nagashima, Steven G. Britt, et al. "Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors." Journal of Cell Biology 206, no. 1 (July 7, 2014): 79–95. http://dx.doi.org/10.1083/jcb.201405020.

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The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

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Keyel, Peter A., Sanjay K. Mishra, Robyn Roth, John E. Heuser, Simon C. Watkins, and Linton M. Traub. "A Single Common Portal for Clathrin-mediated Endocytosis of Distinct Cargo Governed by Cargo-selective Adaptors." Molecular Biology of the Cell 17, no. 10 (October 2006): 4300–4317. http://dx.doi.org/10.1091/mbc.e06-05-0421.

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Sorting of transmembrane cargo into clathrin-coated vesicles requires endocytic adaptors, yet RNA interference (RNAi)-mediated gene silencing of the AP-2 adaptor complex only disrupts internalization of a subset of clathrin-dependent cargo. This suggests alternate clathrin-associated sorting proteins participate in cargo capture at the cell surface, and a provocative recent proposal is that discrete endocytic cargo are sorted into compositionally and functionally distinct clathrin coats. We show here that the FXNPXY-type internalization signal within cytosolic domain of the LDL receptor is recognized redundantly by two phosphotyrosine-binding domain proteins, Dab2 and ARH; diminishing both proteins by RNAi leads to conspicuous LDL receptor accumulation at the cell surface. AP-2–dependent uptake of transferrin ensues relatively normally in the absence of Dab2 and ARH, clearly revealing delegation of sorting operations at the bud site. AP-2, Dab2, ARH, transferrin, and LDL receptors are all present within the vast majority of clathrin structures at the surface, challenging the general existence of specialized clathrin coats for segregated internalization of constitutively internalized cargo. However, Dab2 expression is exceptionally low in hepatocytes, likely accounting for the pathological hypercholesterolemia that accompanies ARH loss.

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Merrifield, C., M. Feldman, and W. Almers. "Dual Wavelength Evanescent Field Microscopy of Exocytosis and Endocytosis in Single Cells." Microscopy and Microanalysis 7, S2 (August 2001): 614–15. http://dx.doi.org/10.1017/s1431927600029147.

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The plasma membrane defines the outer limit of eukaryotic cells and it is here that cells interact with their environment through receptor proteins. Clearly the management of cell surface receptor concentration is critical for normal cellular function, and this is achieved through two key processes, exocytosis and endocytosis. During exocytosis transport vesicles containing both membrane receptors and soluble cargo fuse with the plasma membrane releasing cargo and delivering receptors to the cell surface. During clathrin mediated endocytosis receptors destined for internalisation become concentrated in clathrin coated pits before coat invagination and membrane fission. Since transferrin receptors are recycled and undergo numerous rounds of exocytosis and endocytosis by labeling transferrin receptor with an appropriate fluorophore it is possible to label both exocytic and endocytic organelles in the same cell. We have exploited this phenomenon to visualise the spatial and temporal organisation of constitutive exocytosis and endocytosis at the surface of single cells using dual wavelength evanescent field microscopy.

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Yudowski, Guillermo A., Manojkumar A. Puthenveedu, Anastasia G. Henry, and Mark von Zastrow. "Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway." Molecular Biology of the Cell 20, no. 11 (June 2009): 2774–84. http://dx.doi.org/10.1091/mbc.e08-08-0892.

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Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright “puffs” of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (≈twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

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Ma, Changle, Danielle Hagstrom, Soumi Guha Polley, and Suresh Subramani. "Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5." Journal of Biological Chemistry 288, no. 38 (July 31, 2013): 27220–31. http://dx.doi.org/10.1074/jbc.m113.492694.

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In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1–110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.

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Panté, Nelly, and Michael Kann. "Nuclear Pore Complex Is Able to Transport Macromolecules with Diameters of ∼39 nm." Molecular Biology of the Cell 13, no. 2 (February 2002): 425–34. http://dx.doi.org/10.1091/mbc.01-06-0308.

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Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or “cargoes.” Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.

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Sisodia, S. S. "BIOMEDICINE: A Cargo Receptor Mystery APParently Solved?" Science 295, no. 5556 (February 1, 2002): 805–7. http://dx.doi.org/10.1126/science.1069661.

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Mackmull, Marie‐Therese, Bernd Klaus, Ivonne Heinze, Manopriya Chokkalingam, Andreas Beyer, Robert B. Russell, Alessandro Ori, and Martin Beck. "Landscape of nuclear transport receptor cargo specificity." Molecular Systems Biology 13, no. 12 (December 2017): 962. http://dx.doi.org/10.15252/msb.20177608.

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Lill, Pascal, Tobias Hansen, Daniel Wendscheck, Bjoern Udo Klink, Tomasz Jeziorek, Dimitrios Vismpas, Jonas Miehling, et al. "Towards the molecular architecture of the peroxisomal receptor docking complex." Proceedings of the National Academy of Sciences 117, no. 52 (December 15, 2020): 33216–24. http://dx.doi.org/10.1073/pnas.2009502117.

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Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

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Saraogi, Ishu, David Akopian, and Shu-ou Shan. "Regulation of cargo recognition, commitment, and unloading drives cotranslational protein targeting." Journal of Cell Biology 205, no. 5 (June 9, 2014): 693–706. http://dx.doi.org/10.1083/jcb.201311028.

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Efficient and accurate protein localization is essential to cells and requires protein-targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway. Real-time measurement of cargo transfer from the targeting to translocation machinery revealed multiple factors that drive this event, including GTPase rearrangement in the SRP–SR complex, stepwise displacement of SRP from the ribosome and signal sequence by SecYEG, and elongation of the nascent polypeptide. Our results elucidate how active and sequential regulation of the SRP–cargo interaction drives efficient and faithful protein targeting.

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de Marcos Lousa, Carine, and Jurgen Denecke. "Lysosomal and vacuolar sorting: not so different after all!" Biochemical Society Transactions 44, no. 3 (June 9, 2016): 891–97. http://dx.doi.org/10.1042/bst20160050.

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Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms.

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Mettlen, Marcel, Dinah Loerke, Defne Yarar, Gaudenz Danuser, and Sandra L. Schmid. "Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis." Journal of Cell Biology 188, no. 6 (March 15, 2010): 919–33. http://dx.doi.org/10.1083/jcb.200908078.

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Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself. One of the possible sources of the observed heterogeneous dynamics of clathrin-coated pits (CCPs) might be the different cargo content. Consistent with this, we show that CCP size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH. In Dab2-mCherry–expressing cells, varying LDLR expression leads to a progressive increase in CCP size and to the appearance of nonterminal endocytic events. In LDLR and ARH-mCherry–expressing cells in addition to an increase in CCP size, turnover of abortive CCPs increases, and the rate of CCP maturation decreases. Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs.

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Oostdyk, Luke T., Michael J. McConnell, and Bryce M. Paschal. "Characterization of the Importin-β binding domain in nuclear import receptor KPNA7." Biochemical Journal 476, no. 21 (November 15, 2019): 3413–34. http://dx.doi.org/10.1042/bcj20190717.

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The KPNA family of mammalian nuclear import receptors are encoded by seven genes that generate isoforms with 42–86% identity. KPNA isoforms have the same protein architecture and share the functional property of nuclear localization signal (NLS) recognition, however, the tissue and developmental expression patterns of these receptors raise the question of whether subtle differences in KPNA isoforms might be important in specific biological contexts. Here, we show that KPNA7, an isoform with expression mostly limited to early development, can bind Importin-β (Imp-β) in the absence of NLS cargo. This result contrasts with Imp-β interactions with other KPNA family members, where affinity is regulated by NLS cargo as part of a cooperative binding mechanism. The Imp-β binding (IBB) domain, which is highly conserved in all KPNA family members, generally serves to occlude the NLS binding groove and maintain the receptor in an auto-inhibited ‘closed’ state prior to NLS contact. Cooperative binding of NLS cargo and Imp-β to KPNA results in an ‘open'state. Characterization of KPNA2–KPNA7 chimeric proteins suggests that features of both the IBB domain and the core structure of the receptor contribute to the extent of IBB domain accessibility for Imp-β binding, which likely reflects an ‘open’ state. We also provide evidence that KPNA7 maintains an open-state in the nucleus. We speculate that KPNA7 could function within the nucleus by interacting with NLS-containing proteins.

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Aksu, Metin, Tino Pleiner, Samir Karaca, Christin Kappert, Heinz-Jürgen Dehne, Katharina Seibel, Henning Urlaub, Markus T. Bohnsack, and Dirk Görlich. "Xpo7 is a broad-spectrum exportin and a nuclear import receptor." Journal of Cell Biology 217, no. 7 (May 10, 2018): 2329–40. http://dx.doi.org/10.1083/jcb.201712013.

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Exportins bind cargo molecules in a RanGTP-dependent manner inside nuclei and transport them through nuclear pores to the cytoplasm. CRM1/Xpo1 is the best-characterized exportin because specific inhibitors such as leptomycin B allow straightforward cargo validations in vivo. The analysis of other exportins lagged far behind, foremost because no such inhibitors had been available for them. In this study, we explored the cargo spectrum of exportin 7/Xpo7 in depth and identified not only ∼200 potential export cargoes but also, surprisingly, ∼30 nuclear import substrates. Moreover, we developed anti-Xpo7 nanobodies that acutely block Xpo7 function when transfected into cultured cells. The inhibition is pathway specific, mislocalizes export cargoes of Xpo7 to the nucleus and import substrates to the cytoplasm, and allowed validation of numerous tested cargo candidates. This establishes Xpo7 as a broad-spectrum bidirectional transporter and paves the way for a much deeper analysis of exportin and importin function in the future.

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Keiser, Kristofer J., and Charles Barlowe. "Molecular dissection of the Erv41-Erv46 retrograde receptor reveals a conserved cysteine-rich region in Erv46 required for retrieval activity." Molecular Biology of the Cell 31, no. 3 (February 1, 2020): 209–20. http://dx.doi.org/10.1091/mbc.e19-08-0484.

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The retrograde cargo receptor Erv41-Erv46 retrieves non–KDEL-bearing ER resident proteins to maintain ER homeostasis. This study defines a conserved cysteine-rich element in Erv46 that is required for retrieval activity and cargo binding. We propose that disulfides within the cysteine-rich region regulate the hydrophobic cargo binding site.

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Zellner, Susanne, and Christian Behrends. "Autophagosome content profiling reveals receptor-specific cargo candidates." Autophagy 17, no. 5 (April 5, 2021): 1281–83. http://dx.doi.org/10.1080/15548627.2021.1909410.

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Zhang, Yan, and Zhixiang Chen. "Broad and Complex Roles of NBR1-Mediated Selective Autophagy in Plant Stress Responses." Cells 9, no. 12 (November 30, 2020): 2562. http://dx.doi.org/10.3390/cells9122562.

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Selective autophagy is a highly regulated degradation pathway for the removal of specific damaged or unwanted cellular components and organelles such as protein aggregates. Cargo selectivity in selective autophagy relies on the action of cargo receptors and adaptors. In mammalian cells, two structurally related proteins p62 and NBR1 act as cargo receptors for selective autophagy of ubiquitinated proteins including aggregation-prone proteins in aggrephagy. Plant NBR1 is the structural and functional homolog of mammalian p62 and NBR1. Since its first reports almost ten years ago, plant NBR1 has been well established to function as a cargo receptor for selective autophagy of stress-induced protein aggregates and play an important role in plant responses to a broad spectrum of stress conditions including heat, salt and drought. Over the past several years, important progress has been made in the discovery of specific cargo proteins of plant NBR1 and their roles in the regulation of plant heat stress memory, plant-viral interaction and special protein secretion. There is also new evidence for a possible role of NBR1 in stress-induced pexophagy, sulfur nutrient responses and abscisic acid signaling. In this review, we summarize these progresses and discuss the potential significance of NBR1-mediated selective autophagy in broad plant responses to both biotic and abiotic stresses.

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Baker, Alison, Thomas Lanyon Hogg, and Stuart L. Warriner. "Peroxisome protein import: a complex journey." Biochemical Society Transactions 44, no. 3 (June 9, 2016): 783–89. http://dx.doi.org/10.1042/bst20160036.

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The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes.

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Platta, Harald W., Fouzi El Magraoui, Bastian E. Bäumer, Daniel Schlee, Wolfgang Girzalsky, and Ralf Erdmann. "Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import." Molecular and Cellular Biology 29, no. 20 (August 17, 2009): 5505–16. http://dx.doi.org/10.1128/mcb.00388-09.

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ABSTRACT The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.

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Sato, Ken, and Akihiko Nakano. "Oligomerization of a Cargo Receptor Directs Protein Sorting into COPII-coated Transport Vesicles." Molecular Biology of the Cell 14, no. 7 (July 2003): 3055–63. http://dx.doi.org/10.1091/mbc.e03-02-0115.

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Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII). The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors. Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Exit of Emp46p from the ER was saturable and dependent on the expression level of Emp47p. Emp46p binding to Emp47p occurs in the ER through the coiled-coil region in the luminal domains of both Emp47p and Emp46p, and dissociation occurs in the Golgi. Further, this coiled-coil region is also required for Emp47p to form an oligomeric complex of itself in the ER, which is essential for exit of Emp47p from the ER. Our results suggest that Emp47p is a receptor protein for Emp46p that allows for the selective transport of this protein, and this event involves receptor oligomerization.

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Barr, Francis A., Christian Preisinger, Robert Kopajtich, and Roman Körner. "Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus." Journal of Cell Biology 155, no. 6 (December 10, 2001): 885–92. http://dx.doi.org/10.1083/jcb.200108102.

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The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.

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Behrends, Christian, and Simone Fulda. "Receptor Proteins in Selective Autophagy." International Journal of Cell Biology 2012 (2012): 1–9. http://dx.doi.org/10.1155/2012/673290.

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Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy.

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Appenzeller-Herzog, Christian, Beat Nyfeler, Peter Burkhard, Inigo Santamaria, Carlos Lopez-Otin, and Hans-Peter Hauri. "Carbohydrate- and Conformation-dependent Cargo Capture for ER-Exit." Molecular Biology of the Cell 16, no. 3 (March 2005): 1258–67. http://dx.doi.org/10.1091/mbc.e04-08-0708.

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Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide β-hairpin loop. The motif accounts for lectin ERGIC-53–assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/β-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

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de Wet, Sholto, Andre Du Toit, and Ben Loos. "Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile." Cells 10, no. 1 (January 7, 2021): 95. http://dx.doi.org/10.3390/cells10010095.

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Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

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de Wet, Sholto, Andre Du Toit, and Ben Loos. "Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile." Cells 10, no. 1 (January 7, 2021): 95. http://dx.doi.org/10.3390/cells10010095.

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Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

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Penheiter, Sumedha G., Raman Deep Singh, Claire E. Repellin, Mark C. Wilkes, Maryanne Edens, Philip H. Howe, Richard E. Pagano, and Edward B. Leof. "Type II Transforming Growth Factor-β Receptor Recycling Is Dependent upon the Clathrin Adaptor Protein Dab2." Molecular Biology of the Cell 21, no. 22 (November 15, 2010): 4009–19. http://dx.doi.org/10.1091/mbc.e09-12-1019.

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Transforming growth factor (TGF)-β family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-β receptor recycling as well as Smad2 phosphorylation.

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Liu, Zhigang, Chunlei Zheng, Denise E. Sabatino, and Bin Zhang. "The C Domain Mediates the Export of FVIII from the Endoplasmic Reticulum By Interacting with MCFD2." Blood 128, no. 22 (December 2, 2016): 255. http://dx.doi.org/10.1182/blood.v128.22.255.255.

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Abstract Coagulation factor V (FV) and VIII (FVIII) are large plasma glycoproteins important for hemostasis and thrombosis. After undergoing folding and quality control in the endoplasmic reticulum (ER), FV and FVIII are packaged into COPII (coat protein complex-II) vesicles for transport to the Golgi. We have previously shown that packaging of FV and FVIII into COPII vesicles requires a cargo receptor. This receptor is a Ca2+-dependent protein complex formed by LMAN1 and MCFD2, two proteins that cycle between the ER and the cis-Golgi. Mutations in either LMAN1 or MCFD2 cause the combined deficiency of FV and FVIII, a rare bleeding disorder characterized by the reduction of both FV and FVIII to 5-30% of normal. Cargo receptors are thought to be transmembrane proteins that interact with cargo in the ER lumen and with the COPII coat on the cytoplasmic side of the ER. However, it is not clear how this cargo receptor recognizes and transports cargo. The requirement of a soluble protein (MCFD2) is a unique feature that has not been observed in other known cargo receptors. The basic hypothesis in the receptor-mediated ER-to-Golgi transport model is that specific signals are embedded in cargo proteins that are recognized by their cognate receptors. We previously showed that FV and FVIII interact with both LMAN1 and MCFD2. To identify signals in FV and FVIII that are recognized by MCFD2, we performed immunoprecipitation (IP) of Myc-tagged MCFD2 with a series of Flag-tagged FVIII domain deletion mutants. The light chain of FVIII co-immunoprecipitates with MCFD2, using either anti-Myc or anti-Flag antibodies. The co-IP is mediated through the C domain, not the A3 domain of the light chain. When expressed individually, the C1 domain retains strong co-IP with MCFD2, while the C2 domain shows weaker co-IP with MCFD2. Similarly, only the C domain of FV can co-IP with MCFD2, consistent with the notion that the signal recognized by the LMAN1-MCFD2 complex is a common feature shared by FV and FVIII. To confirm the interaction of the C domain with MCFD2, we used the Bimolecular Fluorescence Complementation assay to detect the interaction in situ. MCFD2 and C domain mutants were separately fused to the N-terminal and the C-terminal halves of the yellow fluorescent protein. When the C1 domain and MCFD2 fusion proteins were co-transfected into HeLa cells, fluorescence signals were detected in live cells, indicating that an interaction between the C1 domain and MCFD2. Transfection of the A3 domain of FVIII produced no fluorescence signals. We further found that the C1 domain of the light chain can significantly increase the heavy chain secretion in pulse-chase experiments. To examine the interaction of MCFD2 with FVIII in vivo, we generated transgenic mice expressing the heavy chain (HC) and light chain (LC) of canine FVIII driven by the liver-specific transthyretin promoter that have been bred into the hemophilia A background. Transgene expression is 30-40 ng/ml for HC and 150-250 ng/ml for LC. We crossed these canine FVIII transgenic mice with our MCFD2 knockout mice and obtained the Mcfd2+/+/HC, Mcfd2-/-/HC, Mcfd2+/+/LC and Mcfd2-/-/LC mouse lines, all of which are in the hemophilia A background. HC and LC levels in plasma were measured by canine FVIII-specific ELISA in 3 month-old mice. Results showed that the plasma LC level was 40% lower in Mcfd2-/- / LC mice than that in Mcfd2+/+/ LC mice (P<0.01), while plasma HC was indistinguishable between Mcfd2+/+/HC and Mcfd2-/-/HC mice. These results indicate that efficient LC secretion is dependent on MCFD2 in vivo, consistent with our cell-based results. In conclusion, our results suggest that MCFD2 recognizes sorting signals located in the C1 and C2 domains of FV and FVIII. The identification of such signals validates the specific cargo and cargo receptor pairing and highlights a direct role of MCFD2 in ER-to-Golgi transport of FV and FVIII. Disclosures No relevant conflicts of interest to declare.

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Caesar, Stefanie, Markus Greiner, and Gabriel Schlenstedt. "Kap120 Functions as a Nuclear Import Receptor for Ribosome Assembly Factor Rpf1 in Yeast." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 3170–80. http://dx.doi.org/10.1128/mcb.26.8.3170-3180.2006.

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ABSTRACT The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin β protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.

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Wijerathna-Yapa, Akila, Elke Stroeher, Ricarda Fenske, Lei Li, Owen Duncan, and A. Harvey Millar. "Proteomics for Autophagy Receptor and Cargo Identification in Plants." Journal of Proteome Research 20, no. 1 (November 26, 2020): 129–38. http://dx.doi.org/10.1021/acs.jproteome.0c00609.

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McNamara, James O., Jeffrey C. Grigston, Hendrika M. A. VanDongen, and Antonius M. J. VanDongen. "Rapid dendritic transport of TGN38, a putative cargo receptor." Molecular Brain Research 127, no. 1-2 (August 2004): 68–78. http://dx.doi.org/10.1016/j.molbrainres.2004.05.013.

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Fromme, J. Christopher. "Membrane Trafficking: Licensing a Cargo Receptor for ER Export." Current Biology 25, no. 2 (January 2015): R67—R68. http://dx.doi.org/10.1016/j.cub.2014.11.051.

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Lévesque, Lyne, Yeou-Cherng Bor, Leah H. Matzat, Li Jin, Stephen Berberoglu, David Rekosh, Marie-Louise Hammarskjöld, and Bryce M. Paschal. "Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex." Molecular Biology of the Cell 17, no. 2 (February 2006): 931–43. http://dx.doi.org/10.1091/mbc.e04-07-0634.

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Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.

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Park, Misoon, Daeseok Lee, Gil-Je Lee, and Inhwan Hwang. "AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole." Journal of Cell Biology 170, no. 5 (August 22, 2005): 757–67. http://dx.doi.org/10.1083/jcb.200504112.

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Organellar proteins are sorted by cargo receptors on the way to their final destination. However, receptors for proteins that are destined for the protein storage vacuole (PSV) are largely unknown. In this study, we investigated the biological role that Arabidopsis thaliana receptor homology region transmembrane domain ring H2 motif protein (AtRMR) 1 plays in protein trafficking to the PSV. AtRMR1 mainly colocalized to the prevacuolar compartment of the PSV, but a minor portion also localized to the Golgi complex. The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion. Coimmunoprecipitation and in vitro binding assays revealed that the lumenal domain of AtRMR1 interacts with the COOH-terminal sorting signal of phaseolin at acidic pH. Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV. Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV.

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Jung, Hyera, Han Nim Lee, Richard S. Marshall, Aaron W. Lomax, Min Ji Yoon, Jimi Kim, Jeong Hun Kim, Richard D. Vierstra, and Taijoon Chung. "Arabidopsis cargo receptor NBR1 mediates selective autophagy of defective proteins." Journal of Experimental Botany 71, no. 1 (September 8, 2019): 73–89. http://dx.doi.org/10.1093/jxb/erz404.

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Teckchandani, Anjali, Erin E. Mulkearns, Timothy W. Randolph, Natalie Toida, and Jonathan A. Cooper. "The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis." Molecular Biology of the Cell 23, no. 15 (August 2012): 2905–16. http://dx.doi.org/10.1091/mbc.e11-12-1007.

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Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain–binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

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Fiuza, Maria, Christine M. Rostosky, Gabrielle T. Parkinson, Alexei M. Bygrave, Nagaraj Halemani, Marcio Baptista, Ira Milosevic, and Jonathan G. Hanley. "PICK1 regulates AMPA receptor endocytosis via direct interactions with AP2 α-appendage and dynamin." Journal of Cell Biology 216, no. 10 (August 30, 2017): 3323–38. http://dx.doi.org/10.1083/jcb.201701034.

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Clathrin-mediated endocytosis (CME) is used to internalize a diverse range of cargo proteins from the cell surface, often in response to specific signals. In neurons, the rapid endocytosis of GluA2-containing AMPA receptors (AMPARs) in response to NMDA receptor (NMDAR) stimulation causes a reduction in synaptic strength and is the central mechanism for long-term depression, which underlies certain forms of learning. The mechanisms that link NMDAR activation to CME of AMPARs remain elusive. PICK1 is a BAR domain protein required for NMDAR-dependent reductions in surface GluA2; however, the molecular mechanisms involved are unclear. In this study, we show that PICK1 makes direct, NMDAR-dependent interactions with the core endocytic proteins AP2 and dynamin. PICK1–AP2 interactions are required for clustering AMPARs at endocytic zones in dendrites in response to NMDAR stimulation and for consequent AMPAR internalization. We further show that PICK1 stimulates dynamin polymerization. We propose that PICK1 is a cargo-specific endocytic accessory protein required for efficient, activity-dependent AMPAR endocytosis.

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Sönnichsen, B., R. Watson, H. Clausen, T. Misteli, and G. Warren. "Sorting by COP I-coated vesicles under interphase and mitotic conditions." Journal of Cell Biology 134, no. 6 (September 15, 1996): 1411–25. http://dx.doi.org/10.1083/jcb.134.6.1411.

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COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

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Schäfer, Antje, Daniela Kerssen, Marten Veenhuis, Wolf-H. Kunau, and Wolfgang Schliebs. "Functional Similarity between the Peroxisomal PTS2 Receptor Binding Protein Pex18p and the N-Terminal Half of the PTS1 Receptor Pex5p." Molecular and Cellular Biology 24, no. 20 (October 15, 2004): 8895–906. http://dx.doi.org/10.1128/mcb.24.20.8895-8906.2004.

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ABSTRACT Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.

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Ramón, Naxhiely Martínez, and Bonnie Bartel. "Interdependence of the Peroxisome-targeting Receptors in Arabidopsis thaliana: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes." Molecular Biology of the Cell 21, no. 7 (April 2010): 1263–71. http://dx.doi.org/10.1091/mbc.e09-08-0672.

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Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.

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Journal articles on the topic 'Cargo receptor'

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