Sip1, an AP-1 Accessory Protein in Fission Yeast,Is Required for Localization of Rho3 GTPase

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3 ⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.     

Role of the Small GTPase Rho3 in Golgi/Endosome trafficking through functional interaction with adaptin in Fission Yeast

Background

We had previously identified the mutant allele of apm1⁺ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast.

Methodology/Principal Findings

In the present study, we isolated rho3⁺, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl⁻ sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl⁻, and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner.

Conclusions/Significance

Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link between the small GTPase Rho and the clathrin-associated adaptor protein-1 in membrane trafficking.     

Cation diffusion facilitator Cis4 is implicated in Golgi membrane trafficking via regulating zinc homeostasis in fission yeast

We screened for mutations that confer sensitivities to the calcineurin inhibitor FK506 and to a high concentration of MgCl₂ and isolated the cis4-1 mutant, an allele of the gene encoding a cation diffusion facilitator (CDF) protein that is structurally related to zinc transporters. Consistently, the addition of extracellular Zn²⁺ suppressed the phenotypes of the cis4 mutant cells. The cis4 mutants and the mutant cells of another CDF-encoding gene SPBC16E9.14c (we named zrg17⁺) shared common and nonadditive zinc-suppressible phenotypes, and Cis4 and Zrg17 physically interacted. Cis4 localized at the cis-Golgi, suggesting that Cis4 is responsible for Zn²⁺ uptake to the cis-Golgi. The cis4 mutant cells showed phenotypes such as weak cell wall and decreased acid phosphatase secretion that are thought to be resulting from impaired membrane trafficking. In addition, the cis4 deletion cells showed synthetic growth defects with all the four membrane-trafficking mutants tested, namely ypt3-i5, ryh1-i6, gdi1-i11, and apm1-1. Interestingly, the addition of extracellular Zn²⁺ significantly suppressed the phenotypes of the ypt3-i5 and apm1-1 mutant cells. These results suggest that Cis4 forms a heteromeric functional complex with Zrg17 and that Cis4 is implicated in Golgi membrane trafficking through the regulation of zinc homeostasis in fission yeast.     

Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.     

Regulation of synaptic activity by snapin‐mediated endolysosomal transport and sorting

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin^−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin^−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca²⁺ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca²⁺ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.     

Leishmania adaptor protein-1 subunits are required for normal lysosome traffic, flagellum biogenesis, lipid homeostasis, and adaptation to temperatures encountered in the mammalian host

The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite Leishmania mexicana mexicana, the AP-1 μ1 and σ1 subunits are not required for growth at 27°C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence. The μ1 and σ1 subunits were localized to late Golgi and endosome membranes of the major parasite stages. Parasite mutants lacking either AP-1 subunit lacked obvious defects in Golgi structure, endocytosis, or exocytic transport. However, these mutants displayed reduced rates of endosome-to-lysosome transport and accumulated fragmented, sterol-rich lysosomes. Defects in flagellum biogenesis were also evident in nondividing promastigote stages, and this phenotype was exacerbated by inhibitors of sterol and sphingolipid biosynthesis. Furthermore, both AP-1 mutants were hypersensitive to elevated temperature and perturbations in membrane lipid composition. The pleiotropic requirements for AP-1 in membrane trafficking and temperature stress responses explain the loss of virulence of these mutants in the mammalian host.     

AP1S3 Mutations Are Associated with Pustular Psoriasis and Impaired Toll-like Receptor 3 Trafficking

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis.     

The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex.

The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae. Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose. These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex. Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose. We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant. We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains. A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4. Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies. These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex. Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.     

The Yeast Adaptor Protein Complex,AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 μ chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Δ vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Δ mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3–dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.The formation of vesicles for transport between membrane-bound organelles requires assembly of coat proteins that are recruited from the cytosol. These proteins direct the sequestration and concentration of cargo as well as invagination of the membrane. One of the best studied classes of coats involved in vesicle budding is comprised of clathrin and its adaptor proteins (APs)¹, AP-1 and AP-2 (Schmid, 1997). In clathrin-mediated vesicle transport the AP complexes play the dual role of cargo selection and recruitment of clathrin to the membrane. These adaptors are heterotetramers containing two large chains (adaptins, α or γ and β), one medium chain (μ), and one small chain (σ). AP-1 (γ, β1, μ1, and σ1) functions in sorting at the TGN, whereas AP-2 (α, β2, μ2, and σ2) is involved in receptor capture at the PM during endocytosis.Although there is a great deal of evidence supporting the involvement of adaptors in clathrin-mediated vesicle budding, recent studies in animal cells have led to the discovery of a novel adaptor-like complex, AP-3, that seems to function independently of clathrin (Newman et al., 1995; Simpson et al., 1996). AP-3 has identical subunit architecture to AP-1 and AP-2, with two adaptin-like subunits (δ and β3), a medium chain (μ3), and a small chain (σ3) (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a , b ). AP-3 antibodies label a perinuclear region, perhaps the TGN, and punctate structures extending to the cell periphery, which may be endosomal compartments (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a ). However, the mammalian AP-3 complex does not colocalize with clathrin or AP-1 and AP-2 adaptors in cells and it does not copurify with brain clathrin-coated vesicles (Newman et al., 1995; Simpson et al., 1996, 1997; Dell''Angelica et al., 1997b ). Clues to the function of AP-3 have come from the discovery that the garnet gene of Drosophila encodes a protein closely related to δ adaptin (Ooi et al., 1997; Simpson et al., 1997). Mutations in garnet cause decreased pigmentation of the eyes and other tissues and a reduced number of pigment granules, which may be lysosome-like organelles (Ooi et al., 1997; Simpson et al., 1997). Thus, AP-3 is proposed to function in clathrin-independent transport between the TGN, endosomes and/or lysosomes, although its exact sorting function is still not known.Over the last several years, yeast homologues of the mammalian adaptor subunits have been identified, allowing for the examination of specific functions of these proteins in a genetically tractable organism. Genes encoding subunits sufficient for at least three complete AP complexes have been identified by sequence homology (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995) or by function (Panek et al., 1997). APL1-APL6 encode large chain/ adaptin-related subunits, APM1-APM4 encode μ-like chains, and APS1-APS3 are genes for σ-related proteins. Apl2p (β), Apl4p (γ), Apm1p (μ1), and Aps1p (σ1) are thought to be subunits of an AP-1–like complex that functions with clathrin at the late Golgi/TGN (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). Mutations in the yeast AP-1 genes enhance the growth and the α-factor processing defects of a temperature sensitive (ts) allele of the clathrin heavy chain gene (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). The latter phenotype is a hallmark of clathrin-deficient yeast, in which late Golgi/ TGN proteins, such as the α-factor processing enzymes Kex2p and dipeptidyl amino peptidase-A (DPAP)-A, are not retained in the late Golgi but escape to the cell surface (Seeger and Payne, 1992b ). To date, no yeast adaptor subunit has been shown to be important for endocytosis, although Apl3p, Apm4p, and Aps2p are most homologous to mammalian AP-2 α, μ2 and σ2, respectively.Recently, a yeast adaptor related to AP-3 of animal cells was described (Panek et al., 1997). It is comprised of Apl5p, Apl6p, Apm3p, and Aps3p, which show preferential homology to mammalian δ, β3, μ3, and σ3, respectively. Mutations in each of these subunits were isolated by their ability to suppress the lethality resulting from loss of function of PM casein kinase 1 encoded by a gene pair, YCK1 and YCK2. Yck activity was found to be required for constitutive endocytosis of the a-factor receptor (Ste3p), and AP-3 subunit mutations partially rescued this internalization defect (Panek et al., 1997). However, the AP complex itself is not necessary for endocytosis, nor is it required for sorting of carboxypeptidase Y (CPY) or retention of late Golgi proteins. Furthermore, unlike disruption of the yeast AP-1 complex, loss of AP-3 function causes no synthetic phenotype in combination with chc1 mutations, suggesting it may function independently of clathrin. Although these data indicated that Apl5p, Apl6p, Apm3p, and Aps3p comprise an AP-3-like adaptor, its precise sorting role was still not known.In this report we describe a genetic approach to determine the function of the yeast AP-3 complex. A colony sectoring screen was performed to identify genes that are essential in the absence of Apm3p, the yeast AP-3 μ chain. Such synthetic lethal screens can be used to identify functional homologues, genes whose proteins function in intersecting or parallel pathways, and genes whose proteins physically interact (Bender and Pringle, 1991). We have cloned the gene for the apm three synthetic lethal mutant, mts1-1, and found it encodes Vps45p, a protein involved in vacuolar protein sorting (Vps; Cowles et al., 1994; Piper et al., 1994). The Vps pathway is defined by >40 complementation groups whose proteins are required for the transport of a number of soluble and membrane-bound proteins, including CPY, protease A (PrA), and carboxypeptidase S (CPS) from the late Golgi/TGN to the vacuole (Stack et al., 1995; Cowles et al., 1997). This pathway is also essential for proper assembly of the vacuolar ATPase (Raymond et al., 1992). However, the type II vacuolar membrane protein alkaline phosphatase (ALP) follows an alternate intracellular pathway to the vacuole (Raymond et al., 1992; Nothwehr et al., 1995; Cowles et al., 1997; Piper et al., 1997). Few vps mutants prevent localization of ALP to the vacuolar membrane and its arrival at the vacuole is not dependent upon transport through the cell surface. The requirement for Apm3p in the absence of Vps45p suggested the possibility that at least one of these routes to the vacuole must be functional for survival and led us to examine ALP sorting in the AP-3 mutants. We show here that yeast AP-3 is essential for the transport of ALP via the alternative pathway to the vacuole.     

Studies on the Roles of Clathrin-Mediated Membrane Trafficking and Zinc Transporter Cis4 in the Transport of GPI-Anchored Proteins in Fission Yeast

We previously identified Cis4, a zinc transporter belonging to the cation diffusion facilitator protein family, and we demonstrated that Cis4 is implicated in Golgi membrane trafficking in fission yeast. Here, we identified three glycosylphosphatidylinositol (GPI)-anchored proteins, namely Ecm33, Aah3, and Gaz2, as multicopy suppressors of the MgCl(2)-sensitive phenotype of cis4-1 mutant. The phenotypes of ecm33, aah3 and gaz2 deletion cells were distinct from each other, and Cis4 overexpression suppressed Δecm33 phenotypes but did not suppress Δaah3 defects. Notably, green fluorescent protein-tagged Ecm33, which was observed at the cell surface in wild-type cells, mostly localized as intracellular dots that are presumed to be the Golgi and endosomes in membrane-trafficking mutants, including Δapm1, ypt3-i5, and chc1-1 mutants. Interestingly, all these membrane-trafficking mutants showed hypersensitivity to BE49385A, an inhibitor of Its8 that is involved in GPI-anchored protein synthesis. Taken together, these results suggest that GPI-anchored proteins are transported through a clathrin-mediated post-Golgi membrane trafficking pathway and that zinc transporter Cis4 may play roles in membrane trafficking of GPI-anchored proteins in fission yeast.     

The β‐subunits of the Snf1 kinase in Saccharomyces cerevisiae,Gal83 and Sip2, but not Sip1, are redundant in glucose derepression and regulation of sterol biosynthesis

The conserved Snf1/AMP‐activated protein kinase family is one of the central components in the nutrient sensing and regulation of the carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic α‐subunit Snf1, a regulatory γ‐subunit Snf4 and one of three possible β‐subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three β‐subunits by analysing all seven possible combinations of β‐subunit deletions together with the reference strain. Previous studies showed that the three β‐subunits are redundant for growth on alternative carbon sources. Here we report that the mutant strain with only SIP1 expressed (sip2Δgal83Δ) could utilize acetate, but neither ethanol nor glycerol, as alternative carbon source. We also showed that Gal83 is the most important isoform not only for the growth on non‐fermentable carbon sources, but also for regulation of ergosterol biosynthetic genes, under glucose‐limited condition. Furthermore, we found that Sip2, but not Sip1, can take over when Gal83 is deleted, but to a lesser extent. However, Sip1 may be sufficient for some other processes such as regulation of the nitrogen metabolism and meiosis.     

A Highlights from MBoC Selection: The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.     

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.     

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.     

Mutation of the Membrane-Associated M1 Protease APM1 Results in Distinct Embryonic and Seedling Developmental Defects in Arabidopsis

Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A–sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA.     

Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1^ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.     

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2

Shiga toxin‐producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A‐subunits block protein synthesis, while the B‐subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B‐subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B‐subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome‐to‐Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface‐exposed loop in STx2B (β4–β5 loop) is required for its endosome‐to‐Golgi trafficking. We previously demonstrated that residues in the corresponding β4–β5 loop of STx1B are required for interaction with GPP130, the STx1B‐specific endosomal receptor, and for endosome‐to‐Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.      

Sip5 interacts with both the Reg1/Glc7 protein phosphatase and the Snf1 protein kinase of Saccharomyces cerevisiae

The Snf1 protein kinase is an essential component of the glucose starvation signalling pathway in Saccharomyces cerevisiae. We have used the two-hybrid system to identify a new protein, Sip5, that interacts with the Snf1 kinase complex in response to glucose limitation. Coimmunoprecipitation studies confirmed the association of Sip5 and Snf1 in cell extracts. We found that Sip5 also interacts strongly with Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase 1 complex, in both two-hybrid and coimmunoprecipitation assays. Previous work showed that Reg1/Glc7 interacts with the Snf1 kinase under glucose-limiting conditions and negatively regulates its activity. Sip5 is the first protein that has been shown to interact with both Snf1 and Reg1/Glc7. Genetic analysis showed that the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5Delta mutant. These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase.     

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.