Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast

Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment.     

A new family of yeast nuclear pore complex proteins

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.     

ATP binding regulates oligomerization and endosome association of RME-1 family proteins

Members of the RME-1/mRme-1/EHD1 protein family have recently been shown to function in the recycling of membrane proteins from recycling endosomes to the plasma membrane. RME-1 family proteins are normally found in close association with recycling endosomes and the vesicles and tubules emanating from these endosomes, consistent with the proposal that these proteins directly participate in endosomal transport. RME-1 family proteins contain a C-terminal EH (eps15 homology) domain thought to be involved in linking RME-1 to other endocytic proteins, a coiled-coil domain thought to be involved in homo-oligomerization and an N-terminal P-loop domain thought to mediate nucleotide binding. In the present study, we show that both Caenorhabditis elegans and mouse RME-1 proteins bind and hydrolyze ATP. No significant GTP binding or hydrolysis was detected. Mutation or deletion of the ATP-binding P-loop prevented RME-1 oligomerization and at the same time dissociated RME-1 from endosomes. In addition, ATP depletion caused RME-1 to lose its endosome association in the cell, resulting in cytosolic localization. Taken together, these results indicate that ATP binding is required for oligomerization of mRme-1/EHD1, which in turn is required for its association with endosomes.     

Annexin A8 regulates late endosome organization and function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.     

A family of yeast proteins mediating bidirectional vacuolar amino acid transport

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.     

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.     

The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome

Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.     

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.     

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.     

Evolution of the Rab family of small GTP-binding proteins.

Rab proteins are small GTP-binding proteins that form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. Here, we have identified the complete Rab families in the Caenorhabditis elegans (29 members), Drosophila melanogaster (29), Homo sapiens (60) and Arabidopsis thaliana (57), and we defined criteria for annotation of this protein family in each organism. We studied sequence conservation patterns and observed that the RabF motifs and the RabSF regions previously described in mammalian Rabs are conserved across species. This is consistent with conserved recognition mechanisms by general regulators and specific effectors. We used phylogenetic analysis and other approaches to reconstruct the multiplication of the Rab family and observed that this family shows a strict phylogeny of function as opposed to a phylogeny of species. Furthermore, we observed that Rabs co-segregating in phylogenetic trees show a pattern of similar cellular localisation and/or function. Therefore, animal and fungi Rab proteins can be grouped in "Rab functional groups" according to their segregating patterns in phylogenetic trees. These functional groups reflect similarity of sequence, localisation and/or function, and may also represent shared ancestry. Rab functional groups can help the understanding of the functional evolution of the Rab family in particular and vesicular transport in general, and may be used to predict general functions for novel Rab sequences.     

Methods of assaying Bcl-2 and Bax family proteins in yeast

Bcl-2 family proteins play an evolutionarily conserved role in regulating the life and death of the cell. Certain proapoptotic members of the Bcl-2 family, Bax and Bak, have intrinsic cytotoxic activities in that they not only induce or sensitize mammalian cells to undergo apoptosis but also display a lethal phenotype when ectopically expressed in two yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. Furthermore, the antiapoptotic Bcl-2 and Bcl-XL proteins can protect yeast against Bax-mediated lethality, suggesting that the death-regulatory functions of these Bcl-2 family proteins are well preserved in yeast. These observations provide the opportunity to study the function of Bcl-2 family proteins in genetically tractable yeast and to apply classical yeast genetics and functional cloning approaches to the dissection of programmed cell death pathway regulated by Bcl-2 family proteins. We describe here methods used in our laboratory to express and to study the functions of Bcl-2 family proteins in both the budding yeast S. cerevisiae and the fission yeast S. pombe.     

A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins.

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.     

Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans

The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.