Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4- ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.     

Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane

Clathrin-mediated vesicular trafficking events underpin the vectorial transfer of macromolecules between several eukaryotic membrane-bound compartments. Classical models for coat operation, focused principally on interactions between clathrin, the heterotetrameric adaptor complexes, and cargo molecules, fail to account for the full complexity of the coat assembly and sorting process. New data reveal that targeting of clathrin adaptor complexes is generally supported by phosphoinositides, that cargo recognition by heterotetrameric adaptors depends on phosphorylation-driven conformational alterations, and that dedicated clathrin-associated sorting proteins (CLASPs) exist to promote the selective trafficking of specific categories of cargo. A host of accessory factors also participate in coat polymerization events, and the independently folded appendage domains that project off the heterotetrameric adaptor core function as recruitment platforms that appear to oversee assembly operations. It is also now clear that focal polymerization of branched actin microfilaments contributes to clathrin-coated vesicle assembly and movement at both plasma membrane and Golgi sites. This improved appreciation of the complex mechanisms governing clathrin-dependent sorting events reveals several common principles of clathrin operation at the Golgi and the plasma membrane.     

The trans-most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins

The intracellular site for the sorting of proteins destined for regulated or constitutive pathways is presently unknown for any one cell. By immunoelectron microscopy, we directly followed the routes taken by a regulated hormone, insulin, and a constitutive protein, hemagglutinin. Both proteins are present in individual Golgi stacks where they appear randomly distributed throughout the cisternae. In contrast, the two proteins do not colocalize outside the Golgi area:insulin is concentrated in dense-core secretory granules, while hemagglutinin is found predominantly in clear 100-300 nm vesicles. These vesicles do not label significantly with an endocytic tracer, indicating that they are exocytic carriers for hemagglutinin. The site at which the two proteins diverge is the clathrin-coated, trans-most cisterna of the Golgi, where the packaging of proinsulin takes place.     

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.     

Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway

We have devised a genetic selection for mutant yeast cells that fail to properly deliver the vacuolar glycoprotein CPY to the lysosome-like vacuole. This has allowed us to identify mutations in eight VPL complementation groups that result in aberrant secretion of up to approximately 90% of the immunoreactive CPY. Other soluble vacuolar proteins are also affected by each vpl mutation, demonstrating that a sorting system for multiple vacuolar proteins exists in yeast. Mislocalized CPY apparently traverses late stages of the secretory pathway, since a vesicle-accumulating sec1 mutation prevents secretion of this protein. Despite the presence of abnormal membrane-enclosed organelles in some of the vpl mutants, maturation and secretion of invertase are not substantially perturbed. Thus vpl mutations define a new class of genes that encode products required for sorting of newly synthesized vacuolar proteins from secretory proteins during their transit through the yeast secretory pathway.     

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.     

Plant RMR proteins: unique vacuolar sorting receptors that couple ligand sorting with membrane internalization

In receptor-mediated sorting of soluble protein ligands in the endomembrane system of eukaryotic cells, three completely different receptor proteins for mammalian (mannose 6-phosphate receptor), yeast (Vps10p) and plant cells (vacuolar sorting receptor; VSR) have in common the features of pH-dependent ligand binding and receptor recycling. In striking contrast, the plant receptor homology-transmembrane-RING-H2 (RMR) proteins serve as sorting receptors to a separate type of vacuole, the protein storage vacuole, but do not recycle, and their trafficking pathway results in their internalization into the destination vacuole. Even though plant RMR proteins share high sequence similarity with the best-characterized mammalian PA-TM-RING family proteins, these two families of proteins appear to play distinctly different roles in plant and animal cells. Thus, this minireview focuses on this unique sorting mechanism and traffic of RMR proteins via dense vesicles in various plant cell types.     

Protein sorting to the vacuolar membrane.

The vacuolar membrane (tonoplast) of plant cells contains a polytopic integral membrane protein with six membrane-spanning domains and cytoplasmically oriented amino-terminal and carboxy-terminal domains. This protein, tonoplast intrinsic protein (TIP), is a member of the membrane intrinsic protein (MIP) family of proteins, a family of channel proteins found in a variety of organisms. In bean seeds, alpha-TIP is synthesized on the rough endoplasmic reticulum and its transport to the tonoplast is mediated by the secretory system. In this study, we report that a polypeptide segment that includes the sixth membrane domain and the cytoplasmic tail of 18 amino acids of alpha-TIP is sufficient to target the reporter protein phosphinotricine acetyltransferase to the tonoplast of stably transformed tobacco cells. To determine if the carboxy-terminal cytoplasmic tail of alpha-TIP contains important tonoplast targeting information, a deletion construct lacking the 15 carboxy-terminal amino acids was introduced for transient expression in tobacco cells; we found that the slightly truncated protein still accumulated in the tonoplast. From these results, we concluded that a transmembrane domain of a tonoplast protein probably contains sufficient information for transport to the tonoplast. Whether such transport occurs by bulk flow or involves specific cellular machinery remains to be determined.     

Molecular sorting of proteins into the cisternal secretory pathway

Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed.     

Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes

The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood on comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognize ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway

Sorting signals for apically destined proteins are highly diverse and can be present within the luminal, membrane-associated, and cytoplasmic domains of these proteins. A subset of apical proteins partition into detergent-resistant membranes, and the association of these proteins with glycolipid-enriched microdomains or lipid rafts may be important for their proper targeting. Recently, we observed that raft-associated and raft-independent apical proteins take different routes to the apical surface of polarized Madin-Darby canine kidney cells (Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007) EMBO J. 26, 3737-3748). Here we reconstituted in vitro the export of raft-associated and raft-independent markers staged intracellularly at 19 degrees C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19 degrees C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.     

Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane

We have established by peptide mapping and immunochemical analysis of purified clathrin assembly protein preparations from bovine brain, that the cluster of components of mol. wt 100-120 kd fall into four classes, which we term alpha, beta, beta' and gamma. The beta and beta' proteins are immunologically related and generate a series of common tryptic peptides. The same criteria reveal no such homologies between the alpha, beta(beta') and gamma polypeptides. The so-called HA-II assembly protein group contains equimolar amounts of alpha and beta class polypeptides, which are shown to interact with each other. In the HA-I group assembly protein complex gamma and beta' class polypeptides form a stoichiometric complex. Immunofluorescence microscopy reveals that the HA-I complex is specifically associated with clathrin-coated membranes in the Golgi region of cultured cells, whereas the HA-II complex appears to be restricted to coated pits on the plasma membrane. The data lead to the tentative conclusion that the clathrin assembly proteins are involved in the recognition of the intracellular targets by uncoated vesicles.     

Ubiquitin ligase adaptors: Regulators of ubiquitylation and endocytosis of plasma membrane proteins

The subcellular localization of plasma membrane proteins, such as receptors and transporters, must be finely tuned so that they can be readily downregulated in response to environmental cues. Some of these membrane proteins are post-translationally modified by conjugation to ubiquitin, which is used as a molecular tag to commit them to the endocytic pathway and promote their subsequent delivery to the lysosomes for degradation. This ubiquitylation step, which is performed by so-called ubiquitin ligases (or E3), appears therefore as a critical event for endocytosis and is subject to many levels of regulation. In this review, we focus on the regulation of cargo ubiquitylation by accessory proteins, or “adaptors”, and discuss the various ways by which they promote the action of ubiquitin ligases toward their specific cargoes. Common features emerge on this mode of regulation, which is present from yeast to human, regardless of the type of ubiquitin ligase in charge of the ubiquitylation. Finally, because these adaptors represent an additional layer of specificity in the ubiquitylation cascade, and can themselves be subject to a complex regulation, they are essential actors in the fine-tuning of endocytosis.     

gamma-Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast.

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

Artificial ubiquitylation is sufficient for sorting of a plasma membrane ATPase to the vacuolar lumen of Arabidopsis cells

Sorting of transmembrane proteins into the inner vesicles of multivesicular bodies for subsequent delivery to the vacuole/lysosome can be induced by attachment of a single ubiquitin or K63-linked ubiquitin chains to the cytosolic portion of the cargo in yeast and mammals. In plants, large efforts have been undertaken to elucidate the mechanisms of vacuolar trafficking of soluble proteins. Sorting of transmembrane proteins, by contrast, is still largely unexplored. As a proof of principle, that ubiquitin is involved in vacuolar sorting in plants we show that a translational fusion of a single ubiquitin to the Arabidopsis plasma membrane ATPase PMA-EGFP is sufficient to induce its endocytosis and sorting into the vacuolar lumen. Sorting of the artificial reporter is not dependent on ubiquitin chain formation, but involves ubiquitin's hydrophobic patch and can be inhibited by coexpression of a dominant-negative version of the ESCRT (endosomal sorting complex required for transport) related protein AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1). Our results suggest that ubiquitin can in principle act as vacuolar sorting signal in plants.