CLN5 is a soluble lysosomal protein with unknown function. Mutations in CLN5 lead to neuronal ceroid lipofuscinosis, a group of inherited neurodegenerative disorders that mainly affect children. CLN5 has eight potential N-glycosylation sites based on the Asn-X-Thr/Ser consensus sequence. Through site-directed mutagenesis of individual asparagine residues to glutamine on each of the N-glycosylation consensus sites, we showed that all eight putative N-glycosylation sites are utilized in vivo. Additionally, localization studies showed that the lack of N-glycosylation on certain sites (N179, N252, N304, or N320) caused CLN5 retention in the endoplasmic reticulum, indicating that glycosylation is important for protein folding. Interestingly, one particular mutant, N401Q, is mislocalized to the Golgi, suggesting that N401 is not important for protein folding but essential for CLN5 trafficking to the lysosome. Finally, we analyzed several patient mutations in which N-glycosylation is affected. The N192S patient mutant is localized to the lysosome, indicating that this mutant has a functional defect in the lysosome. Our results suggest that there are functional differences in various N-glycosylation sites of CLN5 which affect folding, trafficking, and lysosomal function of CLN5.
The microtubule (MT) “plus end” constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called “MT plus-end tracking proteins” (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa
MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.
The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.
The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.
Saccharomyces cerevisiae is the main microorganism responsible for wine alcoholic fermentation. The oenological phenotypes resulting from fermentation, such as the production of acetic acid, glycerol, and residual sugar concentration are regulated by multiple genes and vary quantitatively between different strain backgrounds. With the aim of identifying the quantitative trait loci (QTLs) that regulate oenological phenotypes, we performed linkage analysis using three crosses between highly diverged S. cerevisiae strains. Segregants from each cross were used as starter cultures for 20-day fermentations, in synthetic wine must, to simulate actual winemaking conditions. Linkage analysis on phenotypes of primary industrial importance resulted in the mapping of 18 QTLs. We tested 18 candidate genes, by reciprocal hemizygosity, for their contribution to the observed phenotypic variation, and validated five genes and the chromosome II right subtelomeric region. We observed that genes involved in mitochondrial metabolism, sugar transport, nitrogen metabolism, and the uncharacterized ORF YJR030W explained most of the phenotypic variation in oenological traits. Furthermore, we experimentally validated an exceptionally strong epistatic interaction resulting in high level of succinic acid between the Sake FLX1 allele and the Wine/European MDH2 allele. Overall, our work demonstrates the complex genetic basis underlying wine traits, including natural allelic variation, antagonistic linked QTLs and complex epistatic interactions between alleles from strains with different evolutionary histories.
Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.
Establishment and maintenance of equilibrium in the fatty acid (FA) composition of phospholipids (PL) requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool) and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS) deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL) synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT) Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one.
Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologues of the mammalian oxysterol-binding protein (Osh1–7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in nonvesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.
cholesterol; cyclodextrin; ergosterol; dehydroergosterol; detergent resistant membrane; endoplasmic reticulum; lipid droplet; lipid transfer protein; Osh protein; oxysterol-binding proteins; non-vesicular transport; plasma membrane; yeast
Trs20p is a subunit of the evolutionarily conserved TRAPP (TRAnsport Protein Particle) complex that mediates various aspects of membrane trafficking. Three TRAPP complexes have been identified in yeast with roles in ER-to-Golgi trafficking, post-Golgi and endosomal-to-Golgi transport and in autophagy. The role of Trs20p, which is essential for viability and a component of all three complexes, and how it might function within each TRAPP complex, has not been clarified to date. To begin to address the role of Trs20p we generated different mutants by random mutagenesis but, surprisingly, no defects were observed in diverse anterograde transport pathways or general secretion in Trs20 temperature-sensitive mutants. Instead, mutation of Trs20 led to defects in endocytic recycling and a block in sporulation/meiosis. The phenotypes of different mutants appear to be separable suggesting that the mutations affect the function of Trs20 in different TRAPP complexes.
Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.
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 MgCl2-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 endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non-vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H+ pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane.
The protein cargo transported by specific types of vesicles largely defines the different secretory trafficking pathways operating within cells. However, mole per mole the most abundant cargo contained within transport vesicles is not protein, but lipid. Taking a “lipid-centric” point-of-view, we examine the importance of lipid signaling, membrane lipid organization and lipid metabolism for vesicle transport during exocytosis in budding yeast. In fact, the essential requirement for some exocytosis regulatory proteins can be bypassed by making simple manipulations of the lipids involved. During polarized exocytosis the sequential steps required to generate post-Golgi vesicles and target them to the plasma membrane (PM) involves the interplay of several types of lipids that are coordinately linked through PI4P metabolism and signaling. In turn, PI4P levels are regulated by PI4P kinases, the Sac1p PI4P phosphatase and the yeast Osh proteins, which are homologs of mammalian oxysterol-binding protein (OSBP). Together these regulators integrate the transitional steps required for vesicle maturation directly through changes in lipid composition and organization.
polarized exocytosis; vesicle transport; lipid metabolism; sterols; PI4P; phosphoinositides; SAC1; oxysterol-binding proteins; Osh proteins; small GTPases
The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.
During mating of Saccharomyces cerevisiae, two
nuclei fuse to produce a single diploid nucleus. Two genes,
KAR7 and KAR8, were previously identified
by mutations that cause defects in nuclear membrane fusion.
KAR7 is allelic to SEC71, a gene involved
in protein translocation into the endoplasmic reticulum. Two other
translocation mutants, sec63-1 and
sec72Δ, also exhibited moderate karyogamy defects.
Membranes from kar7/sec71Δ and
sec72Δ, but not sec63-1, exhibited
reduced membrane fusion in vitro, but only at elevated temperatures.
Genetic interactions between kar7 and
kar5 mutations were suggestive of protein–protein
interactions. Moreover, in sec71 mutants, Kar5p was
absent from the SPB and was not detected by Western blot or
immunoprecipitation of pulse-labeled protein. KAR8 is
allelic to JEMI, encoding an endoplasmic
reticulum resident DnaJ protein required for nuclear fusion.
Overexpression of KAR8/JEM1 (but not
SEC63) strongly suppressed the mating defect of
kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p
for nuclear fusion. Electron microscopy analysis of kar8
mutant zygotes revealed a nuclear fusion defect different from
kar2, kar5, and kar7/sec71
mutants. Analysis of double mutants suggested that Kar5p acts before
Kar8/Jem1p. We propose the existence of a nuclear envelope fusion
chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key
components and Sec71p and Sec72p play auxiliary roles.
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.