Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium Photobacterium profundum SS9 at High Pressure and Low Temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae

The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. Here we conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/Gse and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in class C Vps complex components. We find that Tor1 does not regulate the known function of the class C Vps complex in protein sorting. Instead class C vps mutants fail to recover from rapamycin-induced growth arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more effective than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact class C Vps complex is required to mediate intracellular amino acid homeostasis for efficient Tor signaling.     

A conserved GTPase-containing complex is required for intracellular sorting of the general amino-acid permease in yeast

The Saccharomyces cerevisiae general amino-acid permease, Gap1p, is a model for membrane proteins that are regulated by intracellular sorting according to physiological cues set by the availability of amino acids. Here, we report the identification of a conserved sorting complex for Gap1p, named the GTPase-containing complex for Gap1p sorting in the endosomes (GSE complex), which is required for proper sorting of Gap1p from the late endosome for eventual delivery to the plasma membrane. The complex contains two small GTPases (Gtr1p and Gtr2p) and three other proteins (Ybr077c, Ykr007w and Ltv1p) that are located in the late endosomal membrane. Importantly, Gtr2p interacts with the carboxy (C)-terminal cytosolic domain of Gap1p and a tyrosine-containing motif in this domain is necessary both to bind Gtr2p and to direct sorting of Gap1p to the plasma membrane. Together, these studies provide evidence that the GSE complex has a key role in trafficking Gap1p out of the endosome and may serve as coat proteins in this process.     

Actin filaments play a critical role in vacuolar trafficking at the Golgi complex in plant cells

Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurain-like protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of H+-ATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical role in the trafficking of proteins from the Golgi complex to the central vacuole.     

Sorting signals required for trafficking of the cysteine-rich acidic repetitive transmembrane protein in Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis are restricted to the flagellar pocket (FP). The cysteine-rich acidic repetitive transmembrane (CRAM) protein is located at the FP of Trypanosoma brucei and potentially functions as a receptor or an essential component for lipoprotein uptake. We characterized sorting determinants involved in efficient trafficking of CRAM to and from the FP of T. brucei. Previous studies indicated the presence of signals in the CRAM C terminus, specific for its localization to the FP and for efficient endocytosis (H. Yang, D. G. Russell, B. Zeng, M. Eiki, and M.G.-S. Lee, Mol. Cell. Biol. 20:5149-5163, 2000.) To delineate functional domains of putative sorting signals, we performed a mutagenesis series of the CRAM C terminus. Subcellular localization of CRAM mutants demonstrated that the amino acid sequence between -5 and -14 (referred to as a transport signal) is essential for exporting CRAM from the endoplasmic reticulum to the FP, and mutations of amino acids at -12 (V), -10 (V), or -5 (D) led to retention of CRAM in the endoplasmic reticulum. Comparison of the endocytosis efficiency of CRAM mutants demonstrated that the sequence from amino acid -5 to -23 (referred to as a putative endocytosis signal) is required for efficient endocytosis and overlaps with the transport signal. Apparently the CRAM-derived sorting signal can efficiently interact with the T. brucei micro1 adaptin, and mutations at amino acids essential for the function of the transport signal abolished the interaction of the signal with T. brucei micro1, strengthening the hypothesis of the involvement of the clathrin- and adaptor-dependent pathway in trafficking of CRAM via the FP.     

Mitochondrial DNA of two independent oligomycin-resistant Chinese hamster ovary cell lines contains a single nucleotide change in the ATPase 6 gene

We have determined the nucleotide sequence of the URF A6L and ATPase 6 genes of the mitochondrial DNA of wild-type Chinese hamster ovary (CHO) cells and of two independently isolated, cytoplasmically inherited CHO mutant cell lines that are resistant to oligomycin, an inhibitor of the mitochondrial ATP synthase (ATPase) complex. Comparison of the nucleotide sequences of the mutants with that of their parental cell line revealed a single nucleotide difference, a G-to-A transition at nucleotide 433 of the ATPase 6 gene. This single base pair change predicts a nonconservative amino acid change, with a glutamic acid residue being replaced by a lysine residue at amino acid 145 of the ATPase 6 gene product in the mutants. This glutamic acid residue and several others in the surrounding amino acid sequence are conserved among all species examined to date. Analyses of several of the biochemical properties of the oligomycin-resistant CHO mutants indicate that the glutamic acid residue at position 145 of subunit 6 of the mitochondrial ATP synthase complex is important for the binding of oligomycin to the enzyme complex, but is not essential for proton translocation.     

Amino acids regulate retrieval of the yeast general amino acid permease from the vacuolar targeting pathway

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.     

Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.     

Regulation of intracellular phosphatidylinositol-4-phosphate by the Sac1 lipid phosphatase

Phosphatidylinositol 4-phosphate (PtdIns(4)P) regulates diverse cellular processes, such as actin cytoskeletal organization, Golgi trafficking and vacuolar biogenesis. Synthesis and turnover of PtdIns(4)P is mediated by a set of specific lipid kinases and phosphatases. Here we show that the polyphosphoinositide phosphatase Sac1p has a central role in compartment-specific regulation of PtdIns(4)P. We have found that sac1Delta mutants show pleiotropic, synthetically lethal interactions with mutations in genes required for vacuolar protein sorting (Vps). Disruption of the SAC1 gene also caused a defect in the late endocytic pathway. These trafficking phenotypes correlated with a dramatic accumulation of PtdIns(4)P at vacuolar membranes. In addition, sac1 mutants displayed elevated endoplasmic reticulum PtdIns(4)P. The accumulation of PtdIns(4)P at the endoplasmic reticulum and vacuole and the endocytic defect could be compensated by mutations in the PtdIns 4-kinase Stt4p. Our results indicate that elimination of Sac1p causes accumulation of a Stt4p-specific PtdIns(4)P pool at internal membranes which impairs late endocytic and vacuolar trafficking. We conclude that Sac1p functions in confining PtdIns(4)P-dependent processes to specific intracellular membranes.     

Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae

We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome-nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.     

Single amino acid mutations in the Saccharomyces cerevisiae rhomboid peptidase,Pcp1p,alter mitochondrial morphology

Key to mitochondrial activities is the maintenance of mitochondrial morphology, specifically cristae structures formed by the invagination of the inner membrane that are enriched in proteins of the electron transport chain. In Saccharomyces cerevisiae , these cristae folds are a result of the membrane fusion activities of Mgm1p and the membrane‐bending properties of adenosine triphosphate (ATP) synthase oligomerization. An additional protein linked to mitochondrial morphology is Pcp1p, a serine protease responsible for the proteolytic processing of Mgm1p. Here, we have used hydroxylamine‐based random mutagenesis to identify amino acids important for Pcp1p peptidase activity. Using this approach we have isolated five single amino acid mutants that exhibit respiratory growth defects that correlate with loss of mitochondrial genome stability. Reduced Pcp1p protease activity was confirmed by immunoblotting with the accumulation of improperly processed Mgm1p. Ultra‐structural analysis of mitochondrial morphology in these mutants found a varying degree of defects in cristae organization. However, not all of the mutants presented with decreased ATP synthase complex assembly as determined by blue native polyacrylamide gel electrophoresis. Together, these data suggest that there is a threshold level of processed Mgm1p required to maintain ATP synthase super‐complex assembly and mitochondrial cristae organization.     

Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.     

The cytoplasmic tail of glycoprotein M (gpUL100) expresses trafficking signals required for human cytomegalovirus assembly and replication

The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.