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1.
The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.  相似文献   

2.
Amino acid pools were compared in a constructed diploid strain of Saccharomyces cerevisiae , SKD1, and a closely related strain, SKD2, carrying the slp1 mutation characterized by low pools of lysine and lacking a central vacuole. Cells of SKD2 grew more poorly than SKD1 but took up the same total amount of amino acids from the medium per cell although the profile differed between the two strains. Initially, the total pool was much higher in SKD1 than in SKD2 but the overall relative distribution between cytosol and vacuole was identical and mainly cytosolic even though the composition differed between the two strains. At the end of growth the amino acid concentration had increased and become predominantly vacuolar. Two days later the total pool in SKD1 had declined to the starting level but the intracellular distribution remained identical to that at the end of fermentation. The total concentration of amino acids in SKD2 continued to increase, particularly in the cytosol.  相似文献   

3.
Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.  相似文献   

4.
We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery. alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y. (1989) Biochem. Biophys. Res. Commun. 163, 908-915) but is located on the inner surface of the vacuolar membrane. The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide. Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins. None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated. The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations. These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway. We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.  相似文献   

5.
Examination of the subcellular distribution of nickel in a Ni-resistant strain N08 of Saccharomyces cerevisiae showed that 70% of the nickel is distributed in the vascular fraction, which contains large amounts of histidine. The nickel taken up by cells grown in medium containing a high concentration of histidine was preferentially distributed to the vacuole. Arginine and lysine did not affect the intracellular distribution of Ni. In a Ni-sensitive strain 0605-S6, the distribution of nickel into the vacuole was lower than that observed in strain N08. Strain 0605-S6 exhibited no increase in the histidine content of the vacuolar fraction when grown in a Ni-supplemented medium. The Ni-resistant mechanism appears to involve the sequestration of nickel to the vacuole, and histidine could play an important role in the reduction of free nickel in the vacuole by the formation of histidine-nickel complexes.  相似文献   

6.
The pep4-3 mutation results in a 90–95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.  相似文献   

7.
We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.  相似文献   

8.
VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast. The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library. An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype. This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene. On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified. The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure. Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment. They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72. A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins. These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.  相似文献   

9.
The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.  相似文献   

10.
A E Wurmser  S D Emr 《The EMBO journal》1998,17(17):4930-4942
The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40-50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases.  相似文献   

11.
Studies on a proteinase B mutant of yeast.   总被引:1,自引:0,他引:1  
Yeast mutant lacking proteinase B activity have been isolated [Wolf, D. H. and Ehmann, C. (1978) FEBS Lett. 92, 121--124]. One of these mutants (HP232) is characterized in detail. Absence of the vacuolar localized enzyme is confirmed by checking for proteinase B activity in isolated mutant vacuoles. Defective proteinase B activity segregates 2:2 in meiotic tetrads. The mutation is shown to be recessive. Mutant proteinase B activity is not only absent against the synthetic substrate. Azocoll, but also against the physiological substrate pre-chitin synthetase, cytoplasmic malate dehydrogenase and fructose-1,6-bisphosphatase. The mutant shows normal vegetative growth, a phenomenon not consistent with the idea that proteinase B might be the activating principle of chitin synthetase zymogen in vivo. Fluorescence microscopy shows normal chitin insertion. Enzymes underlying carbon-catabolite inactivation in wild-type cells (a mechanism proposed to be possibly triggered by proteinase B) such as cytoplasmic malate dehydrogenase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase, are inactivated also in the mutant. NADP-dependent glutamate dehydrogenase, which is found to be inactivated in glucose-starved wild-type cells, proceeds normally in the mutant. Mutant cells show more than 40% reduced protein degradation under starvation conditions. Sporulating diploids, homozygous for proteinase B absence, also exhibit an approximately 40% reduced protein degradation as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene. The time of the appearance of the first ascospores of diploid cells, homozygous for proteinase B deficiency, is delayed about 50% and sporulation frequency is reduced to about the same extent as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene.  相似文献   

12.
Summary A Saccharomyces cerevisiae mutant which exhibits a considerably increased cellular lysine pool has been isolated and characterized. Assay of enzymes of the lysine and arginine pathways shows that the mutation harboured by this mutant alters the specific repression of lysine but does not influence the general control of amino acid biosynthesis. Because it is recessive to the wild-type allele and acts pleiotropically on the synthesis of several lysine pathway enzymes, this regulatory mutation has been denominated lys80-1 (or lysR –1). It is believed to affect the synthesis or the structure of a factor which plays a negative role in the control of LYS gene expression.  相似文献   

13.
The rice stunted lemma/palea 1 (slp1) mutant displays a dwarf, shortened panicle length, degenerated lemma and palea, and sterility. A previous study suggested that a missense mutation at the sixth amino acid of the OsSPL16 protein was likely to be responsible for the slp1 mutant phenotype. The current study shows that the overexpression of the wild-type OsSPL16 allele in slp1/slp1 and Slp1/slp1 mutants was unable to convert the slp1 mutant phenotype to normal. However, the introduction of the mutant OsSPL16 allele into a normal rice cultivar led to the slp1 mutant phenotype in transgenic plants. These results indicated that the missense mutation in OsSPL16 creates a neomorphic allele, which affects plant height and the development of the inflorescence and spikelet.  相似文献   

14.
The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.  相似文献   

15.
The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of the his1-his7 mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His(+) strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His(-) strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H(+)-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.  相似文献   

16.
A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.  相似文献   

17.
The Saccharomyces cerevisiae PHO8 gene product, repressible alkaline phosphatase (ALP), is a glycoprotein enzyme that is localized to the yeast vacuole (lysosome). Using antibodies raised against synthetic peptides corresponding to two distinct hydrophilic sequences in ALP, we have been able to examine the biosynthesis, sorting and processing of this protein. ALP is synthesized as an inactive precursor containing a C-terminal propeptide that is cleaved from the protein in a PEP4-dependent manner. The precursor and mature protein are anchored in the membrane by an N-terminal hydrophobic domain that also appears to function as an uncleaved internal signal sequence. ALP has the topology of a type-II integral membrane protein containing a short basic N-terminal cytoplasmic tail that is accessible to exogenous protease when associated both with the endoplasmic reticulum and the vacuole. Similar to the soluble vacuolar hydrolases carboxypeptidase Y (CPY) and proteinase A (PrA), ALP transits through the early stages of the secretory pathway prior to vacuolar delivery. Two observations indicate, however, that ALP is localized to the vacuole by a mechanism which is in part different from that used by CPY and PrA: (i) maturation of proALP, which is indicative of vacuolar delivery, is less sensitive than CPY and PrA to the defects exhibited by certain of the vacuolar protein sorting (vps) mutants; and (ii) maturation of proALP proceeds normally in the presence of a potent vacuolar ATPase inhibitor, bafilomycin A1, which is known to block vacuole acidification and leads to the mis-sorting and secretion of precursor forms of CPY and PrA. These results indicate that ALP will be a useful model protein for studies of membrane protein sorting in yeast.  相似文献   

18.
Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.  相似文献   

19.
We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.  相似文献   

20.
We have studied the structure, biosynthesis, intracellular routing, and vacuolar localization of carboxypeptidase ysCS in the yeast Saccharomyces cerevisiae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with different electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77- and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins accumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS precursor forms. Correct proteolytic cleavage of the precursor forms was performed using purified proteinase yscB in vitro. Sec61, sec18, and sec7 mutants, conditionally defective in the secretory pathway, accumulate carboxypeptidase yscS precursor protein. Thus the carboxypeptidase yscS precursor molecules are delivered to the vacuole in a membrane bound form via the secretory pathway. After assembly into the vacuolar membrane, proteinase yscB presumably cleaves the precursor molecules to release soluble carboxypeptidase yscS forms into the lumen of the vacuole. The proposed mechanism is different from the delivery mechanism found for the other soluble vacuolar hydrolases in yeast.  相似文献   

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