Hse1, a component of the yeast Hrs-STAM ubiquitin-sorting complex, associates with ubiquitin peptidases and a ligase to control sorting efficiency into multivesicular bodies

Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.     

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH- terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.     

Vps27 recruits ESCRT machinery to endosomes during MVB sorting

Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.     

Direct binding to Rsp5p regulates ubiquitination-independent vacuolar transport of Sna3p

The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.     

Tom40, the import channel of the mitochondrial outer membrane,plays an active role in sorting imported proteins

The Translocase of the Outer Mitochondrial membrane (TOM complex) is centred on a channel, created by Tom40, serving as the only means of entry for proteins into the mitochondrion. Proteins destined for internal mitochondrial compartments interact subsequently with one of the two distinct protein Translocases of the Inner Mitochondrial membrane (TIM23 and TIM54 complexes) or follow specialized paths into the intermembrane space. To investigate the sorting of precursor proteins to these various sub-mitochondrial compartments, we created a library of tom40 mutants and screened for alleles selectively corrupt in protein sorting. One of the tom40 mutants, tom40-97, carries a single point mutation (W(243)R) resulting in an ineffective transfer of precursors to the TIM23 complex. There is no defect on transfer of precursors to the TIM54 complex or insertion of proteins into the outer membrane. The Tom40 channel is not a passive pore, but plays an active role in protein sorting for all sub-mitochondrial locations.     

K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway

A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus monoubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.     

Hrs and STAM Function Synergistically to Bind Ubiquitin-Modified Cargoes In Vitro

The turnover of integral membrane proteins requires a specialized transport pathway mediated by components of the endosomal sorting complex required for transport (ESCRT) machinery. In most cases, entry into this pathway requires that cargoes undergo ubiquitin-modification, thereby facilitating their sequestration on endosomal membranes by specific, ubiquitin-binding ESCRT subunits. However, requirements underlying initial cargo recognition of mono-ubiquitinated cargos remain poorly defined. In this study, we determine the capability of each ESCRT complex that harbors a ubiquitin-binding domain to bind a reconstituted integral membrane cargo (VAMP2), which has been covalently linked to mono-ubiquitin. We demonstrate that ESCRT-0, but not ESCRT-I or ESCRT-II, is able to associate stably with the mono-ubiquitinated cargo within a lipid bilayer. Moreover, we show that the ubiquitin-binding domains in both Hrs and STAM must be intact to enable cargo binding. These results indicate that the two subunits of ESCRT-0 function together to bind and sequester cargoes for downstream sorting into intralumenal vesicles.     

Receptor downregulation and multivesicular-body sorting

The sorting of proteins into the inner vesicles of multivesicular bodies is required for many key cellular processes, which range from the downregulation of activated signalling receptors to the proper stimulation of the immune response. Recent advances in our understanding of the multivesicular-body sorting pathway have resulted from the identification of ubiquitin as a signal for the efficient sorting of proteins into this transport route, and from the discovery of components of the sorting and regulatory machinery that directs this complex process.     

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C- peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus- dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.     

Biogenesis of the multifunctional lipid droplet: Lipids,proteins, and sites

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca²⁺ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca²⁺ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca²⁺ influx and secretory cargo sorting.     

Ammonium chloride alters secretory protein sorting within the maturing exocrine storage compartment

Addition of 20 mM ammonium chloride during in vitro chase incubation of [35S]methionine pulse-labeled parotid tissue does not perturb the magnitude or radiochemical composition of secretion stimulated by isoproterenol. An apparent inhibition of stimulated output of radiolabeled secretory proteins that was observed when ammonium chloride was added immediately postpulse (but not at later time points prior to stimulation) could be accounted for by slowdown in Golgi transit of exocrine secretory protein at a stage prior to completion of terminal glycosylation. Thus, ammonium chloride does not block entry of newly synthesized secretory proteins into the secretagogue-releasable storage granule compartment. By contrast, ammonium chloride increases the output and substantially alters the relative composition of newly synthesized protein in unstimulated secretion. The latter effects could be assigned to stages of intracellular transport that normally occur at chase times greater than 60 min postpulse and thus are focused within the maturing acinar storage granule. Notably, the compositional alterations cannot reflect the preferential exocytosis of immature granules. Taken together, these results suggest that the sorting of exocrine secretory proteins into the secretagogue-regulated pathway may not involve positive selection by a pH-based process initiated in a pregranule compartment. Rather, unstimulated secretion may arise by a negative sorting (or exclusion) process that occurs during compaction of proteins for storage within maturing granules and that is perturbed by weak base addition. Sorted (or excluded) proteins would appear to follow the vesicular (nongranular) secretory pathway that originates in maturing granules (von Zastrow, M., and Castle, J.D. (1987) J. Cell Biol. 105, 2675-2684).     

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.     

Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.     

Basic mechanisms of secretion: sorting into the regulated secretory pathway.

Targeting proteins to their correct cellular location is crucial for their biological function. In neuroendocrine cells, proteins can be secreted by either the constitutive or the regulated secretory pathways but the mechanism(s) whereby proteins are sorted into either pathway is unclear. In this review we discuss the possibility that sorting is either an active process occurring at the level of the trans-Golgi network, or that sorting occurs passively in the immature granules, The possible involvement of protein-lipid interactions in the sorting process is also raised.     

Two short sequences from amaranth 11S globulin are sufficient to target green fluorescent protein and beta-glucuronidase to vacuoles in Arabidopsis cells.

Vacuolar sorting of seed storage proteins is a very complex process since several sorting pathways and interactions among proteins of different classes have been reported. In addition, although the C-terminus of several 7S proteins is important for vacuolar delivery, other signals seem also to be involved in this process. In this work, the ability of two sequences of the Amaranthus hypochondriacus 11S globulin (amaranthin) to target reporter proteins to vacuoles was studied. We show that the C-terminal pentapeptide (KISIA) and the GNIFRGF internal sequence fused at the C terminal region of genes encoding secretory versions of green fluorescent protein (GFP) and GFP-beta-glucuronidase (GFP-GUS) were sufficient to redirect these reporter proteins to the vacuole of Arabidopsis cells. According to the three-dimensional structure of 7S and 11S storage globulins, this internal vacuolar sorting sequence corresponds to the alpha helical region involved in trimer formation, and is conserved within these families. In addition, these sequences were able to interact in vitro, in a calcium dependent manner, with the sunflower vacuolar sorting receptor homolog to pea BP-80/AtVSR1/pumpkin PV72. This work shows for the first time the role of a short internal sequence conserved among 7S and 11S proteins in vacuolar sorting.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Export of PfSBP1 to the Plasmodium falciparum Maurer's Clefts

The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P.   falciparum -infected erythrocytes.     

Cis-elements of protein transport to the plant vacuoles

Vacuolar proteins are synthesized and translocated into the endoplasmic reticulum and transported to the vacuoles through the secretory pathway. Three different types of vacuolar sorting signals have been identified, carried by N- or C-terminal propeptides or internal sequences. These signals are needed to target proteins to the different types of vacuoles that can coexist in a single plant cell. A conserved motif (NPIXL or NPIR) was identified within N-terminal propeptides, but can also function in a C-terminal propeptide and targets proteins in a receptor-mediated manner to a lytic vacuole. Binding to a family of putative sorting receptors for sequence-specific vacuolar sorting signals has been used as an assay to identify further peptides with other binding motifs. No motif was found in C-terminal sorting sequences, which need an accessible terminus, suggesting that they are recognized from the end by a still unknown receptor. The phosphatidylinositol kinase inhibitor wortmannin differentially affects sorting mediated by these two sorting sequences, suggesting different sorting mechanisms. Less is known about sorting mediated by internal protein sequences, which do not contain the conserved motif identified in N-terminal propeptides and by function by aggregation, leading to transport by coat-less dense vesicles to protein storage vacuoles. Even less is known about the sorting of tonoplast proteins, for which several sorting systems will also be needed.     

Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway

Degradation of various membrane proteins in the lumen of the vacuole/lysosome requires their prior sorting into the multivesicular body (MVB) pathway. In this process, ubiquitin serves as a sorting signal for most cargoes. The yeast ubiquitin hydrolase Doa4 acts late in the MVB pathway. It's role is to catalyze deubiquitination of cargo proteins prior to their sorting into the endosomal vesicles. This step rescues ubiquitin from degradation in the vacuole/lysosome, enabling it to be recycled. Accordingly, the level of monomeric ubiquitin is typically reduced in doa4 mutants. Although MVB sorting of cargo proteins is also impaired in doa4 mutants, the question of whether this defect is due solely to Doa4's role in maintaining a normal pool of ubiquitin in the cell remains open. We here show that the requirement of Doa4 for correct MVB sorting of the endocytic cargo general amino acid permease and of the biosynthetic cargo carboxypeptidase S are not because of the role of Doa4 in ubiquitin recycling. This suggests a direct role of Doa4 in MVB sorting and we show that this role depends on Doa4's catalytic activity. We propose that deubiquitination by Doa4 of cargo proteins and/or some components of the MVB sorting machinery is essential to correct sorting of cargoes into the MVB pathway.     

Clathrin-independent endocytosis: A cargo-centric view

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.