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.     

The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase.

Two polynucleotide-dependent ATPases, 95 and 181 kDa in size, have been purified to near homogeneity from cell-free extracts of Schizosaccharomyces pombe. Despite their size differences, their biochemical properties were strikingly similar. Both enzymes were capable of unwinding RNA and DNA duplexes in keeping with their ability to hydrolyze ATP in the presence of either ribo- or deoxyribopolynucleotide. In addition, they were capable of unwinding DNA/RNA or RNA/DNA hybrid duplexes and translocated in the 5' to 3' direction. These results strongly indicate that they are closely related to each other. Determination of the partial amino acid sequence of the 95-kDa enzyme revealed that it is encoded by the sen1(+)() gene, an S. pombe homologue of yeast SEN1, a protein essential for the processing of small nucleolar RNA, transfer RNA, and ribosomal RNA. The molecular weight of the S. pombe Sen1 protein (SpSen1p) predicted from the sen1(+)() open reading frame was 192.5 kDa, suggesting that the 181-kDa enzyme is likely to be a full-length protein, whereas the 95-kDa polypeptide has arisen by proteolysis. In accord with this possibility, polyclonal antibodies specific to the C-terminal region of sen1(+)() cross-reacted with both 95- and 181-kDa polypeptides. We discuss the biochemical activities associated with SpSen1p and their relevance to the apparently divergent functions ascribed to the yeast Sen1 protein in RNA metabolism.     

Characterization of cDNA encoding mouse homolog of fission yeast dhp1+ gene: structural and functional conservation.

The dhp1+ gene of Schizosaccharomyces pombe is a homolog of Saccharomyces cerevisiae HKE1/RAT1/TAP1 gene that is involved in RNA metabolism such as RNA trafficking and RNA synthesis. dhp1+ is also related to S. cerevisiae DST2 (SEP1) that encodes a DNA strand exchange protein required for sporulation and homologous recombination in S.cerevisiae. We isolated several clones of Dhm1, a mouse homolog of dhp1+, from mouse spermatocyte cDNA library and determined its nucleotide sequence. The Dhm1 gene consists of an open reading frame predicting a protein with 947 amino acids and molecular weight of 107,955. Northern blot analysis revealed that Dhm1 is transcribed at high level in testis, liver and kidney. The predicted product of Dhm1 (Dhm1p) has a significant homology with Dhp1p, Hke1p/Rat1p/Tap1p and Dst2p. In particular, Dhm1p, Dhp1p and Hke1p/Rat1p/Tap1p share strong similarity at the two regions of their N- and C-terminal parts. The Dhm1 gene on a multicopy plasmid rescued the temperature-sensitivity of dhp1ts and lethality of dhp1 null mutation, suggesting that Dhm1 is a mouse homolog of S.pombe dhp1+ and functions similarly in mouse as dhp1+.     

The SpGAR1 gene of Schizosaccharomyces pombe encodes the functional homologue of the snoRNP protein GAR1 of Saccharomyces cerevisiae.

GAR1 is a nucleolar protein which is associated with small nucleolar RNAs (snoRNAs) and which is required for pre-ribosomal RNA processing. In Saccharomyces cerevisiae, the GAR1 gene is essential for cell viability. We have cloned and sequenced the GAR1 gene from the distantly related yeast Schizosaccharomyces pombe. The SpGAR1 gene, which contains two small introns, codes for a 194 amino-acid protein of 20 kDa. A protein sequence comparison indicates that SpGAR1 is 65% identical to ScGAR1. Anti-ScGAR1 antibodies recognize SpGAR1, emphasizing the structural conservation of the protein. Immunostaining of S.pombe cells with these antibodies reveals that SpGAR1 is localized in the nucleolus, as is the case in S.cerevisiae. Moreover, SpGAR1 can substitute for GAR1 in S.cerevisiae, indicating that the two proteins are functionally equivalent. These results suggest a parallel evolutionary conservation of proteins and RNAs with which GAR1 interacts in mediating its pre-rRNA processing and viability functions. After fibrillarin, GAR1 is the second protein of the snoRNPs shown to have been conserved throughout evolution.     

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.     

Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism

We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.     

Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain.     

The Prp1(+) Gene Required for Pre-mRNA Splicing in Schizosaccharomyces Pombe Encodes a Protein That Contains Tpr Motifs and Is Similar to Prp6p of Budding Yeast

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)(+) RNA transport. The prp1(+) gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1(+) and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1(+) gene was found to be identical with the zer1(+) gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)(+) RNA nuclear export, in addition to pre-mRNA splicing.     

Mutation of a conserved glycine residue modifies the vanadate sensitivity of the plasma membrane H+-ATPase from Schizosaccharomyces pombe

The structural gene pma+1 for the H+-ATPase from the fission yeast Schizosaccharomyces pombe has been isolated and sequenced. The intron-less gene encodes for a protein of Mr = 99,769 which is 75% homologous to those of Saccharomyces cerevisiae and Neurospora crassa. The S. pombe pma+1 gene complements not only S. pombe pma-1-1 but also S. cerevisiae pma-1-4 mutants selected for in vitro vanadate-resistant ATPase activity. The sequence of the S. pombe mutant pma-1-1 allele reveals that the glycine residue 268, which is perfectly conserved in the transduction domain of all animal and fungal transport ATPases sequenced so far, is modified into an aspartate residue by the mutation. Replacement of glycine 268 by aspartate has been monitored by the appearance of a new PvuI restriction site in the mutant DNA. Mitotic cosegregation has been observed between the PvuI site and vanadate-resistant ATPase activity in a growing population of S. pombe transformants.     

Mmd1p, a novel, conserved protein essential for normal mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe

The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe. In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures. Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37 degrees C. Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of alpha2-tubulin, display synthetic defects in growth and mitochondrial distribution. The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein. The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms. Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S. cerevisiae gene complemented the S. pombe mutation. Mmd1p was localized to the cytosol. Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

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.     

Amiloride uptake and toxicity in fission yeast are caused by the pyridoxine transporter encoded by bsu1+ (car1+)

Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1+ by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1+ (for vitamin B6 uptake) to replace car1+. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1+ is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.