The Exomer Coat Complex Transports Fus1p to the Plasma Membrane via a Novel Plasma Membrane Sorting Signal in Yeast

Sorting of transmembrane cargo proteins to different cellular compartments is mediated by sorting signals that are recognized by coat proteins involved in vesicle biogenesis. We have identified a sorting signal in the yeast cell fusion protein Fus1p that is required for its transport from the trans-Golgi compartment to the plasma membrane. Transport of Fus1p from the trans-Golgi to the cell surface is dependent on Chs5p, a component of the multisubunit exomer complex. We show that Fus1p transport is also dependent on the exomer components Bch1p and Bud7p. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Fus1p localization to the cell surface in the absence of exomer, possibly by promoting an alternate, exomer-independent route of transport. Mutation of an IXTPK sequence in the cytosolic tail of Fus1p abolishes its physical interaction with Chs5p, results in mislocalization of Fus1p, and therefore causes a cell fusion defect. These defects are suppressed by disruption of AP-1. We suggest that IXTPK comprises a novel sorting signal that is recognized and bound by exomer leading to the capture of Fus1p into coated vesicles en route to the cell surface.     

AP-1A and AP-3A lysosomal sorting functions

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans -Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans -Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans -Golgi network-to-endosome transport and endosome-to- trans -Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized γ-ear containing, ARF binding protein) adaptor proteins in trans -Golgi network to endosome protein transport has brought into question the long-discussed trans -Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.     

A Highlights from MBoC Selection: Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles

Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane–missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN.     

The complex interactions of Chs5p,the ChAPs,and the cargo Chs3p

The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p and the ChAPs family of proteins (Chs6p, Bud7p, Bch1p, and Bch2p), which are believed to act as cargo receptors. In particular, Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p, we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Because these roles are conserved among the ChAPs, the TPRs are interchangeable among different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. Although the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export.     

Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.     

Cargo-sorting signals promote polymerization of adaptor protein-1 in an Arf-1.GTP-independent manner

Adaptor protein-1 (AP-1) is recruited onto the trans-Golgi network via binding to Arf-1·GTP, cargo-sorting signals and phosphoinositides, where it orchestrates the assembly of clathrin-coated vesicular carriers that transport cargo molecules to endosomes. Here we show that cytosolic AP-1 polymerizes when recruited onto enriched Golgi membranes and liposomes containing covalently attached cargo-sorting signal peptides. Incubation of cytosolic or purified AP-1 with soluble sorting signal peptides also resulted in AP-1 polymerization, showing that Arf-1·GTP and membranes are not required for this process. We propose that cargo-induced polymerization of AP-1 contributes to stabilization of the coat complex in the formation of clathrin-coated buds.     

The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins

In yeast, certain resident trans-Golgi network (TGN) proteins achieve steady-state localization by cycling through late endosomes. Here, we show that chitin synthase III (Chs3p), an enzyme involved in the assembly of the cell wall at the mother-bud junction, populates an intracellular reservoir that is maintained by a cycle of transport between the TGN and early endosomes. Traffic of Chs3p from the TGN/early endosome to the cell surface requires CHS5 and CHS6, mutant alleles of which trap Chs3p in the TGN/early endosome. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Chs3p transport to the plasma membrane. Similarly, in AP-1 deficient cells, the resident TGN/early endosome syntaxin, Tlg1p, is missorted. We propose that clathrin and AP-1 act to recycle Chs3p and Tlg1p from the early endosome to the TGN.     

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.     

The polarized epithelia-specific mu 1B-adaptin complements mu 1A-deficiency in fibroblasts

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The µ1-adaptin subunit of the complex exists as the ubiquitous µ1A and the polarized epithelia-specific µ1B, which are 80% identical. In polarized epithelia, µ1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed µ1B in µ1A-deficient fibroblasts to test for µ1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that µ1A and µ1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.     

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1^ad−/−) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1^ad−/− adipocytes. In addition, in Arfrp1^ad−/− adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.     

Widespread Dysregulation of Peptide Hormone Release in Mice Lacking Adaptor Protein AP-3

The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.     

Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment

Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.     

Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.     

The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.     

Clathrin: Its Role in Receptor-Mediated Vesicular Transport and Specialized Functions in Neurons

Abstract

Clathrin constitutes the coat of vesicles involved in three receptor-mediated intracellular transport pathways; the export of aggregated material from the trans-Golgi network for regulated secretion, the transfer of lysosomal hydrolases from the trans-Golgi network to lysosomes and receptor-mediated endocytosis at the plasma membrane. The clathrin subunits and the other major coat constituents, the adaptor polypeptides, interact in specific ways to build the characteristic polygonal clathrin lattice and to attach the coat to integral membrane receptors. Both clathrin coat assembly and disassembly on the cytoplasmic side of the membrane are multistep processes that are regulated by the coat constituents themselves and by cytosolic proteins and factors. Neurons represent a cell type with distinct morphology and special demands on exocytic and endocytic pathways that requires neuron-specific constituents and modifications of clathrin-coated vesicles.     

Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.     

Recognition of sorting signals by clathrin adaptors

Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans-Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrin-adaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin-associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches. BioEssays 21:558–567, 1999. © 1999 John Wiley & Sons, Inc.     

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.     

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking,and Persistent Stuttering

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.     

Cargo trafficking between endosomes and the trans-Golgi network

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.