Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.     

Isolation and analysis of cDNAs encoding small GTP-binding proteins of Arabidopsis thaliana.

We previously isolated a DNA fragment from Arabidopsis thaliana homologous to the mammalian ras gene and named it ara [Matsui et al., Gene 76 (1989) 313-319]. Screening of cDNA clones homologous to ara in A. thaliana resulted in the isolation of four homologous genes. The products of these genes, ARA-2, ARA-3, ARA-4 and ARA-5, showed conservation of amino acids (aa) in four regions, all of which are present in small GTP-binding proteins, and are important for GTPase/GTP-binding activities. These products were highly homologous to those of the YPT genes of Saccharomyces cerevisiae and the ypt gene of Schizosaccharomyces pombe in the regions around aa 45, which is thought to be the site interacting with effector molecules. The products of these four genes showed characteristic aa sequence at their C termini, Cys-Cys-Xaa-Xaa. Another characteristic of this family is presence of Ser in place of Gly in the first conserved region (Gly12 of mammalian GTP-binding Ras protein).     

The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response.

The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle.     

The ras-related ypt protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPT1 gene.

The YPT1 gene of the yeast Saccharomyces cerevisiae codes for a guanine nucleotide-binding protein which is essential for cell viability. Using as hybridization probe cloned yeast YPT1 gene sequences, we have isolated from cDNA libraries prepared from RNA of mouse F9 and C3H10T1/2 cells several overlapping cDNA clones with identical sequence in the regions of overlap. The cDNAs were derived from a gene, designated ypt1, which codes for a protein of 205 amino acids with 71% homology to the yeast YPT1 gene product. Amino acid sequences typical for guanine nucleotide-binding proteins and characteristic for ypt proteins are perfectly conserved in the mouse ypt1 protein. Two mRNAs of 1600 and 3200 nucleotides, originating from the mouse ypt1 gene and differing in the length of their 3'-non-translated region, were identified in mouse F9 cells and in all mouse tissues examined. A monoclonal antibody specifically recognizing the 23.5-kd yeast YPT1 protein cross-reacted with a protein of identical size on protein blots of mouse, rat, pig, bovine and human cell lines.     

Role of a ras homolog in the life cycle of Schizosaccharomyces pombe

We have analyzed the function of the only ras homolog in S. pombe detectable by Southern blotting, ras1, which is homologous to mammalian ras genes and has been cloned. We have disrupted the ras1 gene and have replaced it with ras1Val17, which corresponds to a transforming variant of mammalian ras. Loss of ras1 activity by disruption results in the complete inability to mate. The cell body of a ras1- strain is extensively deformed, and a ras1-/ras1- diploid sporulates very poorly. Unlike RAS1 and RAS2 of S. cerevisiae, ras1 of S. pombe appears to have no effect on adenylate cyclase activity. This suggests that the target enzymes presumably modulated by ras proteins in signal transduction are not the same for all organisms.     

Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe.

Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.     

Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2.     

Trehalose-6-P synthase is dispensable for growth on glucose but not for spore germination in Schizosaccharomyces pombe.

Trehalose-6-P inhibits hexokinases in Saccharomyces cerevisiae (M. A. Blázquez, R. Lagunas, C. Gancedo, and J. M. Gancedo, FEBS Lett. 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose. We have found that the hexokinase from Schizosaccharomyces pombe is not inhibited by trehalose-6-P even at a concentration of 3 mM. The highest internal concentration of trehalose-6-P that we measured in S. pombe was 0.75 mM after heat shock. We have isolated from S. pombe the tps1+ gene, which is homologous to the Saccharomyces cerevisiae TPS1 gene. The DNA sequence from tps1+ predicts a protein of 479 amino acids with 65% identity with the protein of S. cerevisiae. The tps1+ gene expressed from its own promoter could complement the lack of trehalose-6-P synthase in S. cerevisiae tps1 mutants. The TPS1 gene from S. cerevisiae could also restore trehalose synthesis in S. pombe tps1 mutants. A chromosomal disruption of the tps1+ gene in S. pombe did not have a noticeable effect on growth in glucose, in contrast with the disruption of TPS1 in S. cerevisiae. However, the disruption prevented germination of spores carrying it. The level of an RNA hybridizing with an internal probe of the tps1+ gene reached a maximum after 20 min of heat shock treatment. The results presented support the idea that trehalose-6-P plays a role in the control of glycolysis in S. cerevisiae but not in S. pombe and show that the trehalose pathway has different roles in the two yeast species.     

Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p).

From the budding yeast S. cerevisiae, we have cloned a gene, YPT7, that encodes a GTP-binding protein belonging to the Ypt family of ras-related proteins. The 208 amino acid protein shares identical effector domain and C-terminal sequences with the mammalian Rab7 protein. YPT7 gene disruption did not impair cellular growth at temperatures ranging from 17 degrees C to 37 degrees C. ypt7 null mutants are characterized by highly fragmented vacuoles and differential defects of vacuolar protein transport and maturation. The uptake of alpha factor pheromone by wild-type and Ypt7p-deficient cells was found to be indistinguishable, but in mutant cells lacking Ypt7p, degradation of the endocytosed pheromone was severely inhibited. Our findings suggest a role of Ypt7p in protein transport between endosome-like compartments.     

Genetic analysis of yeast RAS1 and RAS2 genes

We present a genetic analysis of RAS1 and RAS2 of S. cerevisiae, two genes that are highly homologous to mammalian ras genes. By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes. However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth. We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.