Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids

2-Hydroxyfatty acids, constituents of brain cerebrosides and sulfatides, were previously reported to be degraded by an alpha-oxidation system, generating fatty acids shortened by one carbon atom. In the current study we used labeled and unlabeled 2-hydroxyoctadecanoic acid to reinvestigate the degradation of this class of lipids. Both in intact and broken cell systems formate was identified as a main reaction product. Furthermore, the generation of an n-1 aldehyde was demonstrated. In permeabilized rat hepatocytes and liver homogenates, studies on cofactor requirements revealed a dependence on ATP, CoA, Mg(2+), thiamine pyrophosphate, and NAD(+). Together with subcellular fractionation data and studies on recombinant enzymes, this led to the following picture. In a first step, the 2-hydroxyfatty acid is activated to an acyl-CoA; subsequently, the 2-hydroxy fatty acyl-CoA is cleaved by 2-hydroxyphytanoyl-CoA lyase, to formyl-CoA and an n-1 aldehyde. The severe inhibition of formate generation by oxythiamin treatment of intact fibroblasts indicates that cleavage through the thiamine pyrophosphate-dependent 2-hydroxyphytanoyl-CoA lyase is the main pathway for the degradation of 2-hydroxyfatty acids. The latter protein was initially characterized as an essential enzyme in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid. Our findings point to a new role for peroxisomes in mammals, i.e. the breakdown of 2-hydroxyfatty acids, at least the long chain 2-hydroxyfatty acids. Most likely, the more abundant very long chain 2-hydroxyfatty acids are degraded in a similar manner.     

LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans

LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.     

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.     

The STF2p hydrophilin from Saccharomyces cerevisiae is required for dehydration stress tolerance

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein.     

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.     

Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.     

Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.     

Ltv1 is required for efficient nuclear export of the ribosomal small subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Deltaltv1 mutants, as judged by the altered distribution of the 5'-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.     

O-mannosylation is required for degradation of the endoplasmic reticulum-associated degradation substrate Gas1*p via the ubiquitin/proteasome pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, protein O-mannosylation, which is executed by protein O-mannosyltransferases, is essential for a variety of biological processes as well as for conferring solubility to misfolded proteins. To determine if O-mannosylation plays an essential role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, we used a model misfolded protein, Gas1*p. The O-mannose content of Gas1*p, which is transferred by protein O-mannosyltransferases, was higher than that of Gas1p. Both Pmt1p and Pmt2p, which do not transfer O-mannose to correctly folded Gas1p, participated in the O-mannosylation of Gas1*p. Furthermore, in a pmt1 Delta pmt2 Delta double-mutant background, degradation of Gas1*p is altered from a primarily proteasome dependent to a vacuolar protease-dependent pathway. This process is in a manner dependent on a Golgi-to-endosome sorting function of the VPS30 complex II. Collectively, our data suggest that O-mannosylation plays an important role for proteasome-dependent degradation of Gas1*p via the ERAD pathway and when O-mannosylation is insufficient, Gas1*p is degraded in the vacuole. Thus, we propose that O-mannosylation by Pmt1p and Pmt2p might be a key step in the targeting of some misfolded proteins for degradation via the proteasome-dependent ERAD pathway.     

Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2 plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.     

The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily Mal (malate), and its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (I(Cl)) by cytosolic Mal or its immediate metabolite OAA (oxaloacetate). In the present study, we have examined the impact of Mal, OAA and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect I(Cl), but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of ICl by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to co-ordinate organic acid metabolism with I(Cl) through the direct effect of organic acid pool size. The findings of the present study also add perspective to in vivo recordings using acetate-based electrolytes.     

Determinants of Swe1p degradation in Saccharomyces cerevisiae

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.