Academic literature on the topic 'Prevacuolar compartment'

Resident membrane proteins of the trans-Golgi network (TGN) ofSaccharomyces cerevisiae are selectively retrieved from a prevacuolar/late endosomal compartment. Proper cycling of the carboxypeptidase Y receptor Vps10p between the TGN and prevacuolar compartment depends on Vps35p, a hydrophilic peripheral membrane protein. In this study we use a temperature-sensitivevps35 allele to show that loss of Vps35p function rapidly leads to mislocalization of A-ALP, a model TGN membrane protein, to the vacuole. Vps35p is required for the prevacuolar compartment-to-TGN transport of both A-ALP and Vps10p. This was demonstrated by phenotypic analysis of vps35 mutant strains expressing A-ALP mutants lacking either the retrieval or static retention signals and by an assay for prevacuolar compartment-to-TGN transport. A novel vps35 allele was identified that was defective for retrieval of A-ALP but functional for retrieval of Vps10p. Moreover, several other vps35 alleles were identified with the opposite characteristics: they were defective for Vps10p retrieval but near normal for A-ALP localization. These data suggest a model in which distinct structural features within Vps35p are required for associating with the cytosolic domains of each cargo protein during the retrieval process.

The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Δ cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.

Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.

ABSTRACTUsing confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungusNeurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases inN. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.

Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar–endosomal compartment, a process dependent on aromatic amino acid–based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal–dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a “receptor” protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

ABSTRACTDisruption of vacuolar biogenesis in the pathogenic yeastCandida albicanscauses profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomalprevacuolarcompartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of thevps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of bothVPS21andYPT52causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only thevps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in avps21Δ/Δypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact onC. albicansphysiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in thevps21Δ/Δypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form ofC. albicans.

We show that the vacuolar protein sorting gene VPS44is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiaeprotein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that ∼35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1Δ cells, and is secreted from the cell.nhx1Δ cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1Δ cells exhibit phenotypes characteristic of the “class E” group ofvps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12Δ andvps34Δ, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10CtΔp mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.

Thesis (Ph. D.)--University of Oregon, 1999.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-152). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948018.

Further in vivo and in vitro studies using the truncated OsRMR1 proteins from the culture media of transgenic BY-2 cells demonstrated that OsRMR1 functioned as a receptor in transporting vicilin-like storage proteins via specific interaction with their vacuolar sorting determinants. Taken together, the OsRMR1 is a sorting receptor for the PSV pathway that defines a novel organelle as PVC for PSV in rice.
Receptor-mediated protein sorting is one of the mechanisms for transporting soluble proteins to the protein storage vacuoles (PSVs) in plant cells. Members of vacuolar sorting receptor (VSR) family proteins and receptor homology region-transmembrane domain-RING-H2 (RMR) family proteins have been shown to function in mediating the transport of storage proteins to PSVs in plants. However, no prevacuolar compartment (PVC) for the PSV pathway has been identified. In this study, I used a rice RMR protein (OsRMR1) as a probe to study the PSV pathway in rice. Using confocal immunofluorescent and immunogold electron microscopy (EM) with specific OsRMR1 antibodies, I have identified a novel organelle as a PVC for the PSV pathway, because OsRMR1 antibodies labeled the Golgi apparatus, trans-Golgi network (TGN) and the novel organelle in both rice cultured cells and developing rice seeds, as well as the protein body Type II (PBII) in developing rice seeds. This novel organelle is morphologically distinct from the lytic PVC or multivesicular body (MVB).
Shen, Yun.
"May 2008."
Adviser: Liwen Jiang.
Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1428.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (p. 124-139).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Nhx1 est un antiport vacuolaire de Na+/H+ chez la levure Saccharomyces cerevisiae. Nhx1 joue un rôle important dans le maintien de l’homéostasie ionique du cytoplasme de la cellule. En effet, la mutation du gène NHX1 chez la levure nhx1Δ entraîne une perte de l’homéostasie cellulaire quand les cellules sont cultivées dans un milieu de faible osmolarité. Ce travail rapporte pour la première fois, et contrairement à la cellule parentale, que la mutation du gène NHX1 a pour effet une sensibilité du mutant nhx1Δ à une variété des drogues et des agents cationiques et anioniques lorsque les cellules sont cultivées dans un milieu riche. En outre, dans ces conditions de culture, aucune sensibilité n’a été observée chez le mutant nhx1Δ quand les cellules sont traitées avec différentes concentrations de sel. Nous avons aussi démontré que la sensibilité du mutant nhx1Δ aux différents agents ainsi que la sécrétion de l’enzyme carboxypeptidase Y observé chez ce mutant n’ont pas été restauré lorsque les cellules sont cultivées dans des milieux avec différents pH ou avec différentes concentrations de sel. Enfin, une analyse génétique a révélé que le mutant nhx1Δ montre un phénotype distinct d’autres mutants qui ont un défaut dans le trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi quand ces cellules sont traitées avec différents agents. Cette analyse prouve que la sensibilité de nhx1Δ aux différents agents n’est pas liée au trafic entre le compartiment pré-vacuolaire et l’appareil de Golgi.
Nhx1 is an intracellular Na+/H+ exchanger localized to the late endosome in Saccharomyces cerevisiae. It is believed that Nhx1 plays a major role in pH-mediated vesicle trafficking, as nhx1Δ mutant is defective in maintaining the intracellular pH in the vacuoles and cytoplasm when grown in low osmolarity media. In this work, we reported novel drug sensitivities of the nhx1Δ mutant to a range of cationic and anionic agents when cells are grown in rich media. Unlike the low osmolarity media, the nhx1Δ mutant showed no sensitivity to salt. Furthermore, we showed that the drug phenotypes of the nhx1Δ mutant, as well as the secretion of the vacuolar protein carboxypeptidase Y, were not rescued by either altering the pH or salt concentration. Although, amino acid substitution of the phylogenetically conserved residue Glu355 for Ala (E355A) in Nhx1 resulted in sensitivity to genotoxic drug bleomycin, it was not observed for the non-conserved residue Glu371Ala (E371A). Moreover, genetic analysis revealed that the nhx1Δ mutant displayed distinct drug phenotypes in comparison to mutants that are defective in retrograde trafficking from the prevacuole to the late Golgi, excluding the possibility that the drug sensitivity of the nhx1Δ mutant is related to retrograde trafficking.

Both structural and immunogold EM studies have also been carried to identify the storage PVCs in developing tobacco seeds and mungbean cotyledons. Biochemically, storage PVCs in both developing tobacco seeds and mungbean cotyledons were labeled by VSRAt-1, S2 (globulin-like proteins), BiP and DIP antibodies. Structurally, storage PVCs in developing tobacco seeds, sized about 200 nm diameter, contain wavy limiting membrane with electron-dense core and periphery translucent outer layer with internal vesicles. In contrast, storage PVCs in mungbean cotyledon, sized about 400 nm diameter, contain electron-dense and translucent area located adjacent to each other.
Further drug treatments studies demonstrated that the lytic PVCs/MVBs in tobacco BY-2 cells were distinct from the storage PVCs in seed cells. BFA and wortmannin treatments respectively caused the lytic PVCs in tobacco BY-2 cells to become aggregate and vacuolated, whereas the storage PVCs in seed cells remained unchanged in response to treatments of these drugs. Therefore, the storage PVCs in developing seeds are biochemically distinct from the lytic PVCs in tobacco BY-2 cells.
Plant cells contain both lytic vacuole and protein storage vacuole. Prevacuolar compartments (PVCs) are membrane-bounded organelles mediating protein trafficking between the Golgi apparatus and vacuoles in the plant secretory pathways. Multivesicular bodies (MVBs) have recently identified as the lytic PVCs in tobacco BY-2 cells. However, little is known about the dynamics of the lytic PVCs. In addition, the existence and identity of PVCs for protein storage vacuole (termed storage PVCs in this study) remain unknown.
This thesis research addressed two important biological questions: the dynamics of the lytic PVCs and the identity of the storage PVCs in plant cells. Towards this goal, I have demonstrated that the Golgi apparatus and the lytic PVCs, marked by YFP fusion reporters in transgenic tobacco BY-2 cells, have different sensitivity to brefeldin A (BFA) treatments. BFA at high concentrations (50 to 100 microg/mL) caused both YFP-marked Golgi stacks and lytic PVCs to form aggregates in a dosage-dependent and time-dependent manner. Confocal immunofluorescence and immunogold EM studies with specific organelle antibody markers further demonstrated that BFA-induced aggregates derived from the lytic PVCs were distinct from but physically associated with the Golgi aggregates. Thus, the BFA effects on the secretory organelles have been extended to the lytic PVCs.
Tse, Yu Chung.
"September 2007."
Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4521.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 156-164).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Lo Sze Wan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 108-115).
Abstracts in English and Chinese.
Thesis committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract (in English) --- p.vi
Abstract (in Chinese) --- p.viii
Table of content --- p.x
List of tables --- p.xv
List of figures --- p.xvi
List of abbreviations --- p.xix
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1 --- The secretory pathway --- p.2
Chapter 1.1.1 --- Endoplasmic reticulum --- p.2
Chapter 1.1.2 --- Golgi complex --- p.3
Chapter 1.1.3 --- Vacuoles --- p.3
Chapter 1.1.4 --- Prevacuolar compartment --- p.4
Chapter 1.2 --- The secretory pathway in plant cells --- p.5
Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7
Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8
Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10
Chapter 1.3 --- Dynamic studies between organelles --- p.12
Chapter 1.4 --- Objectives of this thesis research --- p.13
Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15
Chapter 2.1 --- Introduction --- p.16
Chapter 2.1.1 --- Putative PVC marker --- p.16
Chapter 2.1.2 --- Golgi marker --- p.17
Chapter 2.1.3 --- Dynamic studies --- p.18
Chapter 2.1.4 --- Cell culture study --- p.18
Chapter 2.2 --- Materials and Methods --- p.21
Chapter 2.2.1 --- Plant material --- p.21
Chapter 2.2.2 --- Construction of fusion reporters --- p.22
Chapter 2.2.2.1 --- Cloning materials --- p.22
Chapter 2.2.2.2 --- Vector preparation --- p.22
Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24
Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28
Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30
Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30
Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30
Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31
Chapter 2.2.4.1 --- Killing curve study --- p.31
Chapter 2.2.4.2 --- Antibiotic selection --- p.32
Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33
Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33
Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35
Chapter 2.2.5.1 --- Confocal imaging --- p.35
Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36
Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36
Chapter 2.2.5.4 --- Western blot analysis --- p.37
Chapter 2.2.5.5 --- Cell culture study --- p.37
Chapter 2.3 --- Results --- p.39
Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39
Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40
Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41
Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45
Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47
Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51
Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53
Chapter 2.4 --- Discussion --- p.59
Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59
Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60
Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62
Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63
Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66
Chapter 3.1 --- Introduction --- p.67
Chapter 3.1.1 --- Wortmannin --- p.69
Chapter 3.1.2 --- Brefeldin A --- p.70
Chapter 3.1.3 --- FM4-64 --- p.71
Chapter 3.2 --- Materials and Methods --- p.73
Chapter 3.2.1 --- Plant material --- p.73
Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73
Chapter 3.2.3 --- Drug treatment studies --- p.74
Chapter 3.2.3.1 --- Wortmannin treatment --- p.74
Chapter 3.2.3.2 --- BFA treatment --- p.75
Chapter 3.2.4 --- FM4-64 uptake study --- p.76
Chapter 3.3 --- Results --- p.78
Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78
Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81
Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83
Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88
Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91
Chapter 3.4 --- Discussion --- p.94
Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94
Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95
Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96
Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98
Chapter Chapter 4 --- Summary and Future Prospects --- p.100
Chapter 4.1 --- Summary --- p.101
Chapter 4.1.1 --- Hypothesis --- p.101
Chapter 4.1.2 --- Development of transgenic cell lines --- p.102
Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103
Chapter 4.2 --- Future prospects --- p.106
Reference --- p.108

Cheung Siu Chung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 86-91).
Abstracts in English and Chinese.
Thesis Committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract --- p.v
摘要 --- p.vii
Table of Contents --- p.viii
List of Tables --- p.xiii
List of Figures --- p.xiv
Lists of Abbreviations --- p.xvii
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- The plant secretory pathways --- p.2
Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3
Chapter 1.1.2 --- VSD and VSR --- p.6
Chapter 1.2 --- Prevacuolar compartments --- p.7
Chapter 1.2.1 --- Lytic PVC --- p.7
Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8
Chapter 1.2.3 --- PVC of PSV --- p.9
Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10
Chapter 1.3 --- Project objectives --- p.11
Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments
Chapter 2.1 --- Introduction --- p.13
Chapter 2.2 --- Materials and Methods --- p.15
Chapter 2.2.1 --- Chemicals --- p.15
Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15
Chapter 2.2.3 --- Bacterial Strains --- p.17
Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17
Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17
Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20
Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22
Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24
Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24
Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26
Chapter 2.2.6 --- Agrobacterium electroporation --- p.30
Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30
Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31
Chapter 2.2.8.1 --- Antibiotic selection --- p.31
Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31
Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32
Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32
Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33
Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33
Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34
Chapter 2.2.9.5 --- Confocal microscopic study --- p.35
Chapter 2.3 --- Results --- p.36
Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36
Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37
Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41
Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44
Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48
Chapter 2.4 --- Discussion --- p.53
Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53
Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53
Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55
Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments
Chapter 3.1 --- Introduction --- p.58
Chapter 3.2 --- Materials and Methods --- p.60
Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60
Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62
Chapter 3.2.2.1 --- Wortmannin treatment --- p.62
Chapter 3.2.2.1.1 --- Dosage effect --- p.62
Chapter 3.2.2.1.2 --- Time-course study --- p.62
Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63
Chapter 3.2.2.1.1 --- Dosage effect --- p.63
Chapter 3.2.2.1.2 --- Time-course study --- p.63
Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64
Chapter 3.2.3.1 --- Wortmannin treatment --- p.64
Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64
Chapter 3.3 --- Results --- p.65
Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65
Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69
Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72
Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75
Chapter 3.4 --- Discussion --- p.77
Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77
Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77
Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78
Chapter Chapter 4 --- Summary and Future Prospects
Chapter 4.1 --- Summary --- p.80
Chapter 4.1.1 --- Hypothesis --- p.80
Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81
Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82
Chapter 4.2 --- Conclusions --- p.84
Chapter 4.3 --- Future prospects --- p.85
References --- p.86

Cui, Yong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 73-84).
Abstracts in English and Chinese.
Thesis/Assessment Committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract --- p.v
摘要 --- p.vi
Table of Contents --- p.vii
List of Figures --- p.xi
List of Supplemental Tables --- p.xiii
List of Abbreviations --- p.xiii
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1 --- The plant secretory and endocytosis pathways --- p.2
Chapter 1.2 --- Rab proteins --- p.4
Chapter 1.2.1 --- Overview of the small GTPases --- p.4
Chapter 1.2.2 --- Function of Rab proteins in Arabidopsis --- p.6
Chapter 1.3 --- Prevacuolar compartments --- p.9
Chapter 1.3.1 --- PVCs in mammalian and yeast cells --- p.9
Chapter 1.3.2 --- PVCs in plant cells --- p.9
Chapter 1.4 --- Vacuolar Sorting Receptors --- p.10
Chapter 1.5 --- Project objectives --- p.10
Chapter CHAPTER 2 --- Early and Late Prevacuolar Compartments in Arabidopsis thaliana Cells --- p.12
Chapter 2.1 --- Introduction --- p.13
Chapter 2.2 --- MATERIALS AND METHODS --- p.19
Chapter 2.2.1 --- Plasmid Construction --- p.19
Chapter 2.2.2 --- Plants materials and growth conditions --- p.19
Chapter 2.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.20
Chapter 2.2.4 --- Confocal imaging studies --- p.21
Chapter 2.3 --- RESULTS --- p.23
Chapter 2.3.1 --- Organelle markers serve as a tool to study biogenesis and turnover of PVCs --- p.23
Chapter 2.3.2 --- AtRab5 and AtRab7 proteins show distinct but closely associated patterns in the PVC-to-Vacuole pathway --- p.26
Chapter 2.3.3 --- AtRab5 and AtRab7 proteins localize on the distinct organellein Arabidopsis thaliana protoplasts --- p.32
Chapter 2.3.4 --- AtRab5 proteins are closely associated with AtRab7 proteins --- p.35
Chapter 2.3.5 --- ARA7-Q69L proteins recruit a SNARE complex onto the enlarged PVCs --- p.37
Chapter 2.4 --- Discussion --- p.40
Chapter 2.4.1 --- PVC dynamics in Arabidopsis cells --- p.40
Chapter 2.4.2 --- AtVSR and its point mutation form defined different stages of PVCs in Arabidopsis thaliana protoplasts --- p.41
Chapter 2.4.3 --- AtRab7 proteins localized on the tonoplast and newly defined late PVCs --- p.41
Chapter CHAPTER 3 --- AtRab7 proteins play a critical role in mediating vacuolar trafficking in Arabidopsis thaliana Cells --- p.43
Chapter 3.1 --- Introduction --- p.44
Chapter 3.2 --- MATERIALS AND METHODS --- p.45
Chapter 3.2.1 --- Plasmid Construction --- p.45
Chapter 3.2.2 --- Plants materials and growth conditions --- p.45
Chapter 3.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.45
Chapter 3.2.4 --- Confocal imaging studies --- p.45
Chapter 3.2.5 --- Drug treatment --- p.46
Chapter 3.3 --- RESULTS --- p.48
Chapter 3.3.1 --- Mutations at GTP-binding motifs and the effector domain affect the subcellular localization of AtRabG3e --- p.48
Chapter 3.3.2 --- "AtRabG3e-T22N induced vacuolation of YFP-ARA7 marked PVCs, which remains separated from ER, Golgi and TGN but colocalizes with early PVC markers" --- p.51
Chapter 3.3.3 --- AtRab7-T22N inhibits vacuolar trafficking of cargo proteins --- p.54
Chapter 3.3.4 --- Wortmannin-induced vacuolation of late PVCs in transgenic plants --- p.57
Chapter 3.4 --- Discussion --- p.59
Chapter 3.4.1 --- The proper targeting of AtRab7 proteins --- p.59
Chapter 3.4.2 --- AtRab5 and AtRab7 proteins are essential for vacuolar protein trafficking --- p.59
Chapter CHAPTER 4 --- Summary and Future Perspectives --- p.61
Chapter 4.1 --- Summary --- p.62
Chapter 4.1.1 --- Localization of AtRab5 and AtRab7 proteins on different populations of PVCs --- p.62
Chapter 4.1.2 --- Functions of AtRab7 proteins in Arabidopsis cells --- p.63
Chapter 4.1.3 --- The Rab conversion maturation model --- p.63
Chapter 4.2 --- Future perspectives --- p.64
References --- p.73

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination and post-germination. Here I test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating and post-germination seeds. It is demonstrated that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean seed germination and post-germination. Immunogold electron microscopy (EM) with VSR antibodies demonstrates that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in Day-1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at Day-3 post-germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating and post-germination seeds. Further immunogold EM studies demonstrate that VSR and aleurain colocalize to MVBs, as well as PSVs in germinating and post-germination seeds. Thus, MVBs in germinating and post-germination seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed, and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination and post-germination.
Storage proteins synthesized during seed development are transported to PSVs for storage. However, relatively little is known about the mechanisms of storage protein transport. A putative VSR-interacting protein termed S2 was identified as mung bean 8S globulin. Thus, I test the hypothesis that VSR proteins may be involved in storage protein transport to PSVs in developing mung bean seeds. Immunogold EM with 52 (8S globulin) antibody demonstrates that transport of 8S globulin to PSVs is Golgi-mediated, involving dense vesicle (DV) and a novel prevacuolar compartment (PVC). The novel PVC consists of storage protein aggregates and small internal vesicles. Immunogold EM with S2 (8S globulin) antibody demonstrates that MVBs contain 8S globulin at early stage of seed development. Further immunogold EM studies demonstrate that VSR and 8S globulin colocalize to DVs and the novel PVCs. In vitro binding study demonstrates that calcium ion can stabilize interaction between VSRs and 8S globulin. Thus, VSR proteins may mediate storage protein transport to PSVs via a novel PVC.
Wang, Junqi.
"March 2007."
Adviser: Jiang Liwen.
Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0052.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 120-131).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

by Tse Yu Chung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 133-138).
Abstracts in English and Chinese.
Thesis Committee --- p.ii
Statement --- p.iii
Acknowledgements --- p.iv
Abstract --- p.v
摘要 --- p.vi
Table of Contents --- p.vii
List of Tables --- p.xi
List of Figures --- p.xii
List of Abbreviations --- p.xv
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1. --- The Plant secretory pathway --- p.2
An overview on the secretory pathway --- p.2
Vesicular pathways and transport vesicles --- p.4
Chapter 2. --- Vacuolar sorting receptors --- p.6
BP-80 and its homologues --- p.6
RMR proteins --- p.7
Chapter 3. --- Prevacuolar compartments --- p.8
PVCs in mammalian and yeast cells --- p.8
PVCs for seed protein storage vacuoles --- p.9
PVCs for lytic vacuoles --- p.11
Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15
Chapter 1. --- Introduction --- p.16
Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16
Chapter 1.2 --- Tobacco BY-2 cells --- p.18
Chapter 1.3 --- Plant prevacuolar compartments --- p.19
Chapter 2. --- Materials and Methods --- p.21
Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21
Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27
Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32
Chapter 2.4 --- Agrobacterium electroporation --- p.33
Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34
Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35
Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43
Chapter 2.9 --- Chemicals --- p.45
Chapter 2.10 --- Primers --- p.45
Chapter 2.11 --- Bacterial strain --- p.46
Chapter 3. --- Results --- p.47
Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47
Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55
Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64
Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66
Chapter 4. --- Discussion --- p.68
Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72
Chapter 1. --- Introduction --- p.73
Chapter 1.1 --- The plant secretory pathway --- p.73
Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74
Chapter 1.3 --- Markers for Lytic PVCs --- p.75
Chapter 2. --- Materials and Methods --- p.77
Chapter 2.1 --- Confocal immunofluorescence studies --- p.77
Chapter 2.2 --- FM4-64 uptake study --- p.79
Chapter 2.3 --- Brefeldin A treatment --- p.79
Chapter 2.4 --- Wortmannin treatment --- p.80
Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82
Chapter 3. --- Results --- p.83
Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83
Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86
Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89
Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99
Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102
Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112
Chapter 4. --- Discussion --- p.114
Chapter Chapter 4 --- Summary and Future Perspectives --- p.123
Chapter 1. --- Summary --- p.124
The hypothesis --- p.124
Development of three transgenic cell lines --- p.125
Distinct organelles were marked by two different YFP reporters --- p.126
The YFP-BP-80 reporter defined the lytic PVCs --- p.126
Response of YFP-marked PVCs to Brefeldin A treatment --- p.127
Response of YFP-marked PVCs to Wortmannin treatment --- p.127
PVCs are mobile organelles in living cells --- p.129
Chapter 2. --- Future perspectives --- p.130
References --- p.133