scholarly journals Vam7p, a SNAP-25-Like Molecule, and Vam3p, a Syntaxin Homolog, Function Together in Yeast Vacuolar Protein Trafficking

1998 ◽  
Vol 18 (9) ◽  
pp. 5308-5319 ◽  
Author(s):  
Trey K. Sato ◽  
Tamara Darsow ◽  
Scott D. Emr
Keyword(s):  
Protein Trafficking ◽  
Protein Transport ◽  
Coiled Coil ◽  
Snare Complex ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Nonpermissive Temperature ◽  
Coiled Coil Domain ◽  
Px Domain ◽  
Vacuolar Protein

ABSTRACT A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiaeidentified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671–18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046–3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele ofVAM7 (vam7tsf ) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7tsf mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination ofvam7tsf cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore,VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40phox (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3tsf mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.

scholarly journals A Role for Tlg1p in the Transport of Proteins within the Golgi Apparatus ofSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2407-2423 ◽  
Author(s):  
John G. S. Coe ◽  
Anthony C. B. Lim ◽  
Jing Xu ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Snare Complex ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Nonpermissive Temperature ◽  
Vacuolar Protein

Members of the syntaxin protein family participate in the docking–fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38°C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast α-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.

scholarly journals New Mutants of Saccharomyces cerevisiae Affected in the Transport of Proteins From the Endoplasmic Reticulum to the Golgi Complex

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 393-406 ◽  
Author(s):  
Linda J Wuestehube ◽  
Rainer Duden ◽  
Arlene Eun ◽  
Susan Hamamoto ◽  
Paul Korn ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Membrane Structures ◽  
Α Subunit ◽  
Complementation Groups ◽  
Vacuolar Protein

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

scholarly journals Characterization of a Novel Yeast SNARE Protein Implicated in Golgi Retrograde Traffic

1997 ◽  
Vol 8 (12) ◽  
pp. 2659-2676 ◽  
Author(s):  
Vladimir V. Lupashin ◽  
Irina D. Pokrovskaya ◽  
James A. McNew ◽  
M. Gerard Waters
Keyword(s):  
Protein Trafficking ◽  
Sequence Similarity ◽  
Integral Membrane Protein ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Snare Protein ◽  
Yeast Viability ◽  
Vacuolar Protein

The protein trafficking machinery of eukaryotic cells is employed for protein secretion and for the localization of resident proteins of the exocytic and endocytic pathways. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs) which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle. A novelSaccharomyces cerevisiae SNARE protein, which has been termed Vti1p, was identified by its sequence similarity to known SNAREs. Vti1p is a predominantly Golgi-localized 25-kDa type II integral membrane protein that is essential for yeast viability. Vti1p can bind Sec17p (yeast SNAP) and enter into a Sec18p (NSF)-sensitive complex with the cis-Golgi t-SNARE Sed5p. This Sed5p/Vti1p complex is distinct from the previously described Sed5p/Sec22p anterograde vesicle docking complex. Depletion of Vti1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the Golgi. Temperature-sensitive mutants of Vti1p show a similar carboxypeptidase Y trafficking defect, but the secretion of invertase and gp400/hsp150 is not significantly affected. The temperature-sensitive vti1 growth defect can be rescued by the overexpression of the v-SNARE, Ykt6p, which physically interacts with Vti1p. We propose that Vti1p, along with Ykt6p and perhaps Sft1p, acts as a retrograde v-SNARE capable of interacting with the cis-Golgi t-SNARE Sed5p.

scholarly journals Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

1990 ◽  
Vol 110 (6) ◽  
pp. 1897-1909 ◽  
Author(s):  
J Nair ◽  
H Müller ◽  
M Peterson ◽  
P Novick
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Coiled Coil ◽  
Protein Product ◽  
Temperature Sensitive ◽  
Vesicular Traffic ◽  
Amino Terminal ◽  
Native Molecular Mass ◽  
Coiled Coil Domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

scholarly journals Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

1999 ◽  
Vol 10 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Miki Tsukada ◽  
Elke Will ◽  
Dieter Gallwitz
Keyword(s):  
Protein Transport ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Golgi Compartment ◽  
Helical Content ◽  
Associated Proteins ◽  
Yeast Genes ◽  
Vacuolar Protein

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations.SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to thecis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

scholarly journals End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

1997 ◽  
Vol 8 (11) ◽  
pp. 2291-2306 ◽  
Author(s):  
A. Wesp ◽  
L. Hicke ◽  
J. Palecek ◽  
R. Lombardi ◽  
T. Aust ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Actin Organization ◽  
Coiled Coil Domain ◽  
Associated Proteins

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) ofABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

scholarly journals Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex

2003 ◽  
Vol 162 (1) ◽  
pp. 125-137 ◽  
Author(s):  
Wei Sun ◽  
Qing Yan ◽  
Thomas A. Vida ◽  
Andrew J. Bean
Keyword(s):  
Coiled Coil ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fission Reactions ◽  
Coiled Coil Domain ◽  
Rat Brain Membranes ◽  
Early Endosomes ◽  
Endosomal Membranes

Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150–206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13–SNAP-25–VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.

scholarly journals Synthetic Genetic Interactions With Temperature-Sensitive Clathrin in Saccharomyces cerevisiae: Roles for Synaptojanin-Like Inp53p and Dynamin-Related Vps1p in Clathrin-Dependent Protein Sorting at the trans-Golgi Network

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 83-97
Author(s):  
Eric S Bensen ◽  
Giancarlo Costaguta ◽  
Gregory S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Protein Sorting ◽  
Genetic Interactions ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Trans Golgi Network ◽  
Vacuolar Protein ◽  
Golgi Network

Abstract Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone α-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in α-factor maturation defects and exacerbated α-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.

scholarly journals Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

2015 ◽  
Vol 112 (6) ◽  
pp. 1886-1891 ◽  
Author(s):  
Caiji Gao ◽  
Xiaohong Zhuang ◽  
Yong Cui ◽  
Xi Fu ◽  
Yilin He ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Protein Transport ◽  
Multivesicular Body ◽  
Organelle Biogenesis ◽  
Functional Roles ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Autophagic Degradation ◽  
Vacuolar Protein ◽  
Vacuole Biogenesis

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

scholarly journals Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast.

1995 ◽  
Vol 129 (2) ◽  
pp. 321-334 ◽  
Author(s):  
J H Stack ◽  
D B DeWald ◽  
K Takegawa ◽  
S D Emr
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Sorting ◽  
Intracellular Localization ◽  
Kinase Domain ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3-kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.

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