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T H Stevens

Publications and source records attributed to T H Stevens.

At least 37 records · Page 2Linked to original sources
PubMed1998

A human homolog can functionally replace the yeast vesicle-associated SNARE Vti1p in two vesicle transport pathways.

Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here we describe a human Vti1p homolog, hVti1. Whereas vti1Delta cells are inviable, expression of hVti1 allows vti1Delta cells to grow at nearly the wild-type growth rate. When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi. Sequence comparisons with a Schizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted alpha-helix. Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins.

Amino Acid Sequence↗
PubMed1998

Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route.

The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.

Biological Transport↗
PubMed1998

Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole.

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.

Biological Transport↗
PubMed1997

V1-situated stalk subunits of the yeast vacuolar proton-translocating ATPase.

The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits. The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane. Additional subunits in both sectors couple these activities. Here we have continued our examination of intermediate subunits primarily associated with the V1 but also implicated in interactions with the V0. Interactions between Vma7p (F) and Vma8p (D) and between Vma4p (E) and Vma10p (G) are described. Although Vma7p and Vma10p have been observed to interact with the V0 sector, our results indicate that these subunits behave primarily as canonical V1 sector subunits. We categorize these four subunits as "stalk" subunits to distinguish them from the known catalytic (A and B) and proton-translocating (c, c', and Vma16p) subunits and to highlight their intermediate nature. Furthermore, we show that the in vivo stability of Vma4p is dependent upon interaction with Vma10p. This may be important in the regulation of assembly, since these two subunits add to the V1 during later stages of V1 assembly. This is the first demonstration of interdependence between ATPase subunits for structural stability.

Fungal Proteins↗
PubMed1997

VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H+-ATPase assembly.

The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment.

Cell Compartmentation↗
PubMed1997

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway.

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

Alkaline Phosphatase↗
PubMed1997

The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p.

Membrane traffic in eukaryotic cells requires that specific v-SNAREs on transport vesicles interact with specific t-SNAREs on target membranes. We identified a novel Saccharomyces cerevisiae v-SNARE (Vti1p) encoded by the essential gene, VTI1. Vti1p interacts with the prevacuolar t-SNARE Pep12p to direct Golgi to prevacuolar traffic. vti1-1 mutant cells missorted and secreted the soluble vacuolar hydrolase carboxypeptidase Y (CPY) rapidly and reversibly when vti1-1 cells were shifted to the restrictive temperature. However, overexpression of Pep12p suppressed the CPY secretion defect exhibited by vti1-1 cells at 36 degrees C. Characterization of a second vti1 mutant, vti1-11, revealed that Vti1p also plays a role in membrane traffic at a cis-Golgi stage. vti1-11 mutant cells displayed a growth defect and accumulated the ER and early Golgi forms of both CPY and the secreted protein invertase at the nonpermissive temperature. Overexpression of the yeast cis-Golgi t-SNARE Sed5p suppressed the accumulation of the ER form of CPY but did not lead to CPY transport to the vacuole in vti1-11 cells. Overexpression of Sed5p allowed growth in the absence of Vti1p. In vitro binding and coimmunoprecipitation studies revealed that Vti1p interacts directly with the two t-SNAREs, Sed5p and Pep12p. These data suggest that Vti1p plays a role in cis-Golgi membrane traffic, which is essential for yeast viability, and a nonessential role in the fusion of Golgi-derived vesicles with the prevacuolar compartment. Therefore, a single v-SNARE can interact functionally with two different t-SNAREs in directing membrane traffic in yeast.

Biological Transport↗
PubMed1997

Identification of a mammalian Golgi Sec1p-like protein, mVps45.

Our understanding of lysosomal biogenesis and general vesicular transport in animal cells has been greatly enhanced by studies of vacuolar biogenesis in yeast. Genetic screens have identified a number of proteins that play direct roles in the proper sorting of vacuolar hydrolases. These include t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Sec1p-like proteins, which have recently been implicated as key regulators of vesicle fusion. In this study we have extended these observations in yeast and have isolated and characterized a novel member of the Sec1p-like family of proteins from mammalian cells, mVps45. mVps45 shares a high level of identity with the Saccharomyces cerevisiae Sec1p-like protein Vps45p that is believed to function with the t-SNARE Pep12p in the fusion of Golgi-derived transport vesicles with a prevacuolar compartment. We found that mVps45 is a ubiquitously expressed peripheral membrane protein that localized to perinuclear Golgi-like and trans-Golgi network compartments in Chinese hamster ovary cells. We found that mVps45 could bind specifically to yeast Pep12p and to the mammalian Pep12p-like protein, syntaxin 6, in vitro.

Amino Acid Sequence↗
PubMed1997

VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase.

The vacuolar membrane H+-ATPase (V-ATPase) of the yeast Saccharomyces cerevisiae is composed of peripheral catalytic (V1) and integral membrane (V0) domains. The 17-kDa proteolipid subunit (VMA3 gene product; Vma3p) is predicted to constitute at least part of the proton translocating pore of V0. Recently, two VMA3 homologues, VMA11 and VMA16 (PPA1), have been identified in yeast, and VMA11 has been shown to be required for the V-ATPase activity. Cells disrupted for the VMA16 gene displayed the same phenotypes as those lacking either Vma3p or Vma11p; the mutant cells lost V-ATPase activity and failed to assemble V-ATPase subunits onto the vacuolar membrane. Epitope-tagged Vma11p and Vma16p were detected on the vacuolar membrane by immunofluorescence microscopy. Density gradient fractionation of the solubilized vacuolar proteins demonstrated that the tagged proteins copurified with the V-ATPase complex. We conclude that Vma11p and Vma16p are essential subunits of the V-ATPase. Vma3p contains a conserved glutamic acid residue (Glu137) whose carboxyl side chain is predicted to be important for proton transport activity. Mutational analysis of Vma11p and Vma16p revealed that both proteins contain a glutamic acid residue (Vma11p Glu145 and Vma16p Glu108) functionally similar to Vma3p Glu137. These residues could only be functionally substituted by an aspartic acid residue, because other mutations we examined inactivated the enzyme activity. Assembly and vacuolar targeting of the enzyme complex was not inhibited by these mutations. These results suggest that the three proteolipid subunits have similar but not redundant functions, each of which is most likely involved in proton transport activity of the enzyme complex. Yeast cells contain V0 and V1 subcomplexes in the vacuolar membrane and in the cytosol, respectively, that can be assembled into the active V0V1 complex in vivo. Surprisingly, loss-of-function mutations of either Vma11p Glu145 or Vma16p Glu108 resulted in a higher degree of assembly of the V1 subunits onto the V0 subcomplex in the vacuolar membrane.

Amino Acid Sequence↗
PubMed1997

Two separate signals act independently to localize a yeast late Golgi membrane protein through a combination of retrieval and retention.

The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue-based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.

Alkaline Phosphatase↗
PubMed1997

Structure, function and regulation of the vacuolar (H+)-ATPase.

The vacuolar (H+)-ATPases (or V-ATPases) function in the acidification of intracellular compartments in eukaryotic cells. The V-ATPases are multisubunit complexes composed of two functional domains. The peripheral V1 domain, a 500-kDa complex responsible for ATP hydrolysis, contains at least eight different subunits of molecular weight 70-13 (subunits A-H). The integral V0 domain, a 250-kDa complex, functions in proton translocation and contains at least five different subunits of molecular weight 100-17 (subunits a-d). Biochemical and genetic analysis has been used to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and coupling of these activities. Several mechanisms have been implicated in the regulation of vacuolar acidification in vivo, including control of pump density, regulation of assembly of V1 and V0 domains, disulfide bond formation, activator or inhibitor proteins, and regulation of counterion conductance. Recent information concerning targeting and regulation of V-ATPases has also been obtained.

Animals↗
PubMed1996

Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases.

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.

Aspartic Acid Endopeptidases↗
PubMed1996

The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its

Alkaline Phosphatase↗
PubMed1996

The VPS8 gene is required for localization and trafficking of the CPY sorting receptor in Saccharomyces cerevisiae.

To better understand the process of protein sorting to the yeast vacuole, the VPS8 gene was identified and characterized. VPS8 encodes a membrane-associated hydrophilic protein of 135 kDa (Vps8p), which is required for the accurate sorting of the vacuolar hydrolase, carboxypeptidase Y (CPY). vps8 mutant cells missort and secrete CPY as well as a second soluble vacuolar hydrolase, proteinase A. In vps8 mutants, several late-Golgi membrane proteins fail to be retained in the Golgi apparatus. The Golgi-localized CPY sorting receptor, Vps10p, is mislocalized to and aberrantly proteolyzed in the vacuole. Based on our findings, we propose that Vps8p is part of a protein complex that associates with Golgi and post-Golgi membranes and functions in the retrieval of Golgi membrane proteins from the prevacuolar compartment.

Amino Acid Sequence↗
PubMed1995

Vma22p is a novel endoplasmic reticulum-associated protein required for assembly of the yeast vacuolar H(+)-ATPase complex.

The Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit complex that can be structurally and functionally divided into peripheral (V1) and integral membrane (V0) sectors. The vma22-1 mutation was isolated in a screen for mutants defective in V-ATPase function vma22 delta cells contain no V-ATPase activity due to a failure to assemble the enzyme complex; V1 subunits accumulate in the cytosol, and the V0 100-kDa subunit is rapidly degraded. Turnover of the 100-kDa integral membrane protein was found to occur in the endoplasmic reticulum (ER) of vma22 delta cells. The product of the VMA22 gene, Vma22p, is a 21-kDa hydrophilic protein that is not a subunit of the V-ATPase but rather is associated with ER membranes. The association of Vma22p with ER membranes was perturbed by mutations in VMA12, a gene that encodes an ER membrane protein (Vma12p) that is also required for V-ATPase assembly. These results indicate that Vma22p, along with Vma21p and Vma12p, form a set of ER proteins required for V-ATPase assembly.

Amino Acid Sequence↗
PubMed1995

VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase required for function and assembly of the enzyme complex.

The isolated Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits. We have identified VMA8, the gene encoding the 32-kDa subunit of the V-ATPase, by 100% match between the sequences of tryptic peptides and the predicted protein sequence of ORF11. The VMA8 gene contains a 768-base pair open reading frame encoding a 256-amino acid protein with a predicted molecular mass of 29,176 Da. Disruption of VMA8 resulted in a mutant exhibiting pH-sensitive growth, slowed growth under all conditions, and an inability to grow on nonfermentable carbon sources. Vacuolar membranes isolated from vma8 delta yeast cells exhibited no V-ATPase activity. Immunoblot analysis of vma8 delta cells revealed normal levels of both V1 and Vo subunits. Whereas the V1 subunits failed to associated with the vacuolar membrane in vma8 delta cells, the Vo polypeptides were transported to and stable in the vacuolar membrane. Density gradient fractionation revealed that Vma8p associated only with the fully assembled V-ATPase and did not associate with a separate lower density Vo subcomplex fraction. Finally, Vma8p was unable to assemble onto the vascular membranes in the absence of other V1 subunits.

Amino Acid Sequence↗
PubMed1995

Protein splicing: self-splicing of genetically mobile elements at the protein level.

Protein splicing is a newly discovered process that is the protein equivalent of RNA splicing. Protein splicing proceeds through a branched protein intermediate, and in vitro studies indicate that the reaction is autocatalytic. The excised 'intein' proteins are site-specific DNA endonucleases that catalyse genetic mobility of their DNA coding sequence by an 'intein homing' mechanism.

Amino Acid Sequence↗