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H R Pelham

Publications and source records attributed to H R Pelham.

At least 19 recordsLinked to original sources
PubMed2001

Traffic through the Golgi apparatus.

The role of vesicles in cargo transport through the Golgi apparatus has been controversial. Large forms of cargo such as protein aggregates are thought to progress through the Golgi stack by a process of cisternal maturation, balanced by a return flow of Golgi resident proteins in COPI-coated vesicles. However, whether this is the primary role of vesicles, or whether they also serve to transport small cargo molecules in a forward direction has been debated. Two papers (Martínez-Menárguez et al., 2001; Mironov et al., 2001, this issue) use sophisticated light and electron microscopy to provide evidence that the vesicular stomatitis virus membrane glycoprotein (VSV G)* is largely excluded from vesicles in vivo, and does not move between cisternae, whereas resident Golgi enzymes freely enter vesicles as predicted by the cisternal maturation model. Both papers conclude that vesicles are likely to play only a minor role in the anterograde transport of cargo through the Golgi apparatus in mammalian tissue culture cells.

COP-Coated Vesicles↗
PubMed2001

An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes.

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome-derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p-Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N-terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p-independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.

Cytoplasmic Vesicles↗
PubMed2001

Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting.

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.

Acid Anhydride Hydrolases↗
PubMed2001

Membrane traffic: how do GGAs fit in with the adaptors?

The AP-1 adaptor complex has been cast as the major player in clathrin coat formation for vesicular transport from the trans-Golgi to the endocytic pathway. But new results on 'GGA' proteins have raised doubts about this paradigm and suggest both a new sorting mechanism and an unexpected complexity in the roles of clathrin.

ADP-Ribosylation Factor 1↗
PubMed2001

SNAREs and the specificity of membrane fusion.

A major problem of intracellular membrane traffic concerns the way in which transport vesicles find and fuse with their target organelles. SNARE proteins are involved in fusion, and their mutual recognition could in principle provide the necessary specificity. Alternatively, the preliminary tethering of vesicles, mediated by peripheral membrane proteins, could hold the key. Previous studies of SNARE complex assembly in solution have suggested little specificity, but recent experiments with yeast SNAREs anchored in liposomes show that their interactions can be highly selective. It is likely that both tethering and SNARE engagement contribute to the accuracy of membrane transport.

Amino Acid Sequence↗
PubMed2000

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins.

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes. By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.

ADP-Ribosylation Factors↗
PubMed2000

Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p.

Cells lacking the GTPase Ypt6p have defects in intracellular traffic and are temperature sensitive. Their growth is severely impaired by additional mutation of IMH1, which encodes a non-essential Golgi-associated coiled-coil protein. A screen for mutants that, like ypt6, specifically impair the growth of imh1 cells led to the identification of RIC1. Ric1p forms a tight complex with a previously uncharacterized protein, Rgp1p. The Ric1p-Rgp1p complex binds Ypt6p in a nucleotide-dependent manner, and purified Ric1p-Rgp1 stimulates guanine nucleotide exchange on Ypt6p in vitro. Deletion of RIC1 or RGP1, like that of YPT6, blocks the recycling of the exocytic SNARE Snc1p from early endosomes to the Golgi and causes temperature-sensitive growth, but this defect can be relieved by overexpression of YPT6. Ric1p largely colocalizes with the late Golgi marker Sec7p. Ypt6p shows a similar distribution, but this is altered when RIC1 or RGP1 is mutated. We infer that the Ric1p-Rgp1p complex serves to activate Ypt6p on Golgi membranes by nucleotide exchange, and that this is required for efficient fusion of endosome-derived vesicles with the Golgi.

Amino Acid Sequence↗
PubMed2000

Psr1p/Psr2p, two plasma membrane phosphatases with an essential DXDX(T/V) motif required for sodium stress response in yeast.

Regulation of intracellular ion concentration is an essential function of all cells. In this study, we report the identification of two previously uncharacterized genes, PSR1 and PSR2, that perform an essential function under conditions of sodium ion stress in the yeast Saccharomyces cerevisiae. Psr1p and Psr2p are highly homologous and were identified through their homology with the endoplasmic reticulum membrane protein Nem1p. Localization and biochemical fractionation studies show that Psr1p is associated with the plasma membrane via a short amino-terminal sequence also present in Psr2p. Growth of the psr1psr2 mutant is severely inhibited under conditions of sodium but not potassium ion or sorbitol stress. This growth defect is due to the inability of the psr1psr2 mutant to properly induce transcription of ENA1/PMR2, the major sodium extrusion pump of yeast cells. We provide genetic evidence that this regulation is independent of the phosphatase calcineurin, previously implicated in the sodium stress response in yeast. We show that Psr1p contains a DXDX(T/V) phosphatase motif essential for its function in vivo and that a Psr1p-PtA fusion purified from yeast extracts exhibits phosphatase activity. Based on these data, we suggest that Psr1p/Psr2p, members of an emerging class of eukaryotic phosphatases, are novel regulators of salt stress response in yeast.

Amino Acid Motifs↗
PubMed2000

Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome-Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

Amino Acid Sequence↗
PubMed2000

Polar transmembrane domains target proteins to the interior of the yeast vacuole.

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.

Amino Acid Sequence↗
PubMed1999

The Croonian Lecture 1999. Intracellular membrane traffic: getting proteins sorted.

The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins move on their way to or from the cell surface. Sorting of proteins within this system is achieved by their selective incorporation into budding vesicles and the specific fusion of these with an appropriate target membrane. Cytosolic coat proteins help to select vesicle contents, while fusion is mediated by membrane proteins termed SNAREs present in both vesicles and target membranes. SNAREs are not the sole determinants of target specificity, but they lie at the heart of the fusion process. The complete set of SNAREs is known in yeast, and analysis of their locations, interactions and functions in vivo gives a comprehensive picture of the traffic routes and the ways in which organelles such as the Golgi apparatus are formed. The principles of protein and lipid sorting revealed by this analysis are likely to apply to a wide variety of eukaryotic cells.

Adenosine Triphosphatases↗
PubMed1999

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.

Alleles↗
PubMed1999

SNAREs and the secretory pathway-lessons from yeast.

SNARE proteins lie at the heart of the membrane fusion events in the secretory and endocytic pathways. Physical interactions between them are thought not only to provide the driving force for bringing membranes together, but also to contribute to the specificity of vesicle targeting. Completion of the yeast genome sequence has allowed the full set of SNAREs to be identified. Characterization of these helps to define the number of distinct compartments and the nature of the transport steps between them, but also shows that SNAREs are by no means the sole determinants of fusion specificity. Evolutionary conservation of SNAREs suggests that despite the differences in scale and morphology, many features of membrane organization are similar in yeast and animal cells. This review summarizes current knowledge of the yeast SNAREs and the picture of the secretory pathway that emerges from such studies.

Animals↗
PubMed1998

SNAREs and membrane fusion in the Golgi apparatus.

Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell. They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs. Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion. NSF is an ATPase which will disrupt complexes composed of different SNAREs. However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane. The essential role of NSF may be to prime SNAREs for a direct role during fusion. The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi. The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar. Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes. Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles. In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue. There are only eight members of the syntaxin family in yeast. Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex. They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi. We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles.

Animals↗
PubMed1998

Identification of amino acids in the binding pocket of the human KDEL receptor.

Retention of soluble proteins in the endoplasmic reticulum is dependent on their interaction with the KDEL (Lys-Asp-Glu-Leu) receptor in the Golgi apparatus and their subsequent retrieval back to the endoplasmic reticulum. We have studied the three-dimensional organization of the human KDEL receptor using site-directed mutagenesis and sulfhydryl-specific labeling. We have identified four amino acid residues, Arg-5, Asp-50, Tyr-162, and Asn-165, which we suggest participate in the formation of the ligand binding pocket. Arg-5 and Asp-50 are shown to be located on the lumenal side of the membrane and are inaccessible from the cytoplasm. In addition, our results strongly support a topology of the KDEL receptor similar to the family of G-protein-coupled receptors with seven transmembrane domains. Furthermore, Asp-50 plays a crucial role in the binding of His/Lys-Asp-Glu-Leu ligands, but is not required for Asp-Asp-Glu-Leu binding, suggesting that this residue forms an ion pair with the positively charged amino acid residue positioned 4 residues from the carboxyl terminus of the ligand.

Amino Acid Sequence↗
PubMed1998

A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion.

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

Adenosine Triphosphatases↗