scholarly journals The Doa4 Deubiquitinating Enzyme Is Functionally Linked to the Vacuolar Protein-sorting and Endocytic Pathways

2000 ◽  
Vol 11 (10) ◽  
pp. 3365-3380 ◽  
Author(s):  
Alexander Y. Amerik ◽  
Jonathan Nowak ◽  
Sowmya Swaminathan ◽  
Mark Hochstrasser
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Sorting ◽  
Late Endosome ◽  
Deubiquitinating Enzyme ◽  
Vacuolar Protein Sorting ◽  
Prevacuolar Compartment ◽  
Green Fluorescent ◽  
Vacuolar Protein ◽  
Class E

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin–proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the clonedDID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4–green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4–green fluorescent protein, like several other Vps factors, concentrates at the late endosome–like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

scholarly journals Vacuolar Protein Sorting Protein 13A, TtVPS13A, Localizes to the Tetrahymena thermophila Phagosome Membrane and Is Required for Efficient Phagocytosis

2011 ◽  
Vol 10 (9) ◽  
pp. 1207-1218 ◽  
Author(s):  
Haresha S. Samaranayake ◽  
Ann E. Cowan ◽  
Lawrence A. Klobutcher
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Genetic Disorders ◽  
Protein Sorting ◽  
Mass Spectrometry Analysis ◽  
Vacuolar Protein Sorting ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Green Fluorescent ◽  
Vacuolar Protein

ABSTRACT Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990–2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.

scholarly journals Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

1992 ◽  
Vol 3 (12) ◽  
pp. 1389-1402 ◽  
Author(s):  
C K Raymond ◽  
I Howald-Stevenson ◽  
C A Vater ◽  
T H Stevens
Keyword(s):  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Complementation Groups ◽  
Prevacuolar Compartment ◽  
Golgi Protein ◽  
Mother Cells ◽  
Vacuolar Protein ◽  
Morphological Phenotype

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.

scholarly journals HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway

2004 ◽  
Vol 168 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Juan Martin-Serrano ◽  
Scott W. Eastman ◽  
Wayne Chung ◽  
Paul D. Bieniasz
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Sorting ◽  
Ubiquitin Ligases ◽  
Dominant Negative ◽  
Specific Class ◽  
Vacuolar Protein Sorting ◽  
Viral Budding ◽  
Virion Release ◽  
Vacuolar Protein ◽  
Class E

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.

scholarly journals Vps10p Cycles between the TGN and the Late Endosome via the Plasma Membrane in Clathrin Mutants

2002 ◽  
Vol 13 (12) ◽  
pp. 4296-4307 ◽  
Author(s):  
Olivier Deloche ◽  
Randy W. Schekman
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Late Endosome ◽  
Coated Vesicles ◽  
Cargo Protein ◽  
Endosomal Membranes ◽  
Green Fluorescent ◽  
Vacuolar Protein

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in achc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport inchc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, inchc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normalVPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.

scholarly journals Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.

1996 ◽  
Vol 7 (6) ◽  
pp. 985-999 ◽  
Author(s):  
S E Rieder ◽  
L M Banta ◽  
K Köhrer ◽  
J M McCaffery ◽  
S D Emr
Keyword(s):  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Intense Staining ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein ◽  
Class E

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease

2002 ◽  
Vol 115 (1) ◽  
pp. 217-226 ◽  
Author(s):  
Christelle Marchal ◽  
Sophie Dupré ◽  
Daniele Urban-Grimal
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Direct Role ◽  
Casein Kinase 1 ◽  
Late Step ◽  
Prevacuolar Compartment ◽  
Green Fluorescent

The modification of yeast uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating transporter endocytosis. Uracil permease is phosphorylated at several serine residues within a well characterized PEST sequence. The phosphorylation of these residues facilitates the ubiquitination and internalization of the permease. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have shown that in casein kinase 1 (CK1)-deficient cells, the permease is poorly phosphorylated, poorly ubiquitinated and that Yck activity may play a direct role in phosphorylating the permease. We show here that CK1-deficient cells accumulated permease that was subjected to endocytosis in an internal compartment on its way to the vacuole. Uracil permease, produced as a fusion protein with green fluorescent protein in CK1-deficient cells, was detected in dots adjacent to the vacuole. These dots probably correspond to the late endosome/prevacuolar compartment because they were partially colocalized with the Pep12p marker. This accumulation was abolished by mutations affecting the adaptor-related complex, AP-3. The CPY and ALP pathways to the vacuole were both unaffected in CK1-deficient cells. Our analysis provides the first evidence that CK1 is important for the delivery of proteins to the vacuole after endocytosis.

scholarly journals Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

2005 ◽  
Vol 71 (8) ◽  
pp. 4856-4861 ◽  
Author(s):  
Mamoru Ohneda ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Cell Polarity ◽  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Ph Response ◽  
Isolation And Characterization ◽  
Vacuolar Protein ◽  
Egfp Fluorescence

ABSTRACT The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

scholarly journals Quick Delivery of Aldehyde Dehydrogenase into Yeast Vacuoles by QRPL Peptide Sequence on Carboxypeptidase Y

2020 ◽  
Author(s):  
Dong Jun Park ◽  
Ngoc-Tu Nguyen ◽  
Ji Hun Kim ◽  
Ngoc-Han Nguyen ◽  
Sunchang Kim ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Aldehyde Dehydrogenase ◽  
Signal Peptide ◽  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Carboxypeptidase Y ◽  
Signal Peptide Sequence ◽  
Green Fluorescent ◽  
Vacuolar Protein ◽  
Vacuolar Targeting

Abstract Background The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. Results The signal peptide sequence QRPL of carboxypeptidase Y (CPY), a vacuolar protein, was inserted inside the green fluorescent protein (GFP) that does not locate in vacuole for vacuolar targeting. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 hour after induction). In addition, aldehyde dehydrogenase 6 (ALD6), a cytosolic protein has engineered the sequence with QRPL to be transported to the vacuole. The aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri . Conclusions In summary, the signal peptide QRPL could be used not only to transport target proteins accurately to vacuole but also to improve the protein activity, as well as to shorten the induction time.

scholarly journals VPS21Controls Entry of Endocytosed and Biosynthetic Proteins into the Yeast Prevacuolar Compartment

2000 ◽  
Vol 11 (2) ◽  
pp. 613-626 ◽  
Author(s):  
Sonja R. Gerrard ◽  
Nia J. Bryant ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Endosomal Compartment ◽  
Early Endosomes ◽  
Strong Homology ◽  
Rab Family ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class DVPS genes include VPS21,PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

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