Advancement through Mitosis requires rae1 Gene Function in Fission Yeast

Growth of the rae1-1 mutant of Schizosaccharomyces pombe at restrictive temperature results in accumulation of poly(A)⁺ RNA in the nucleus and a cell cycle arrest at the G2/M boundary. We demonstrate here that rae1 function is required for a process other than mRNA export which is essential for advancement through mitosis. Cells lacking rae1 function arrest with elevated Cdc2p kinase levels at a step before the formation of a mitotic spindle and without separation of the spindle pole bodies. Rae1p was localized to the nuclear periphery, consistent with a role in nucleocytoplasmic trafficking, which could include protein import. We propose a model where rae1 functions in cell cycle progression through trafficking of proteins required for mitosis. © 1997 John Wiley & Sons, Ltd.     

粟酒裂殖酵母降苹果酸基因克隆及其序列分析

粟酒裂殖酵母（Schizosaccharomyces pombe）由于可降解苹果酸（malo-ethanolic fermentation）而被广泛应用于葡萄酒等果酒的酿造。其生理活性依靠的是苹果酸通透酶（MAEI）和苹果酸酶（MAE2）2个关键酶的作用。以粟酒裂殖酵母基因组DNA为模板，采用聚合酶链式反应（PCR）技术，扩增得到苹果酸通透酶基因（mae1）和苹果酸酶基因（mae2），并插入到pMD—T载体中，转化大肠杆菌得到相应的转化子。测序后分析表明，扩增的mae1基因与mae2基因序列与已报道的序列同源性均为99％，mae1基因编码的氨基酸序列中有2个与报道不同；mae2基因编码的氨基酸中有3个与报道不同。     

Identification and Preliminary Characterization of p31, a New PSTAIRE-related Protein in Fission Yeast

One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is the so-called PSTAIRE motif. Western blots of fission yeast cytosolic extracts using a monoclonal antibody against the PSTAIRE peptide revealed two bands at 34 kDa (p34^cdc2) and 31 kDa (p31). Polyclonal antibodies to the C-terminus of p34^cdc2 or to the full-length protein recognized the 34 kDa band but not p31. Overexpression of the cdc2⁺ gene resulted in the increase of the 34 kDa band but not p31. Like p34 the level of p31 revealed no obvious cell cycle regulation but the protein was present in spores where p34^cdc2 was barely detectable. p31 expression was unaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34^cdc2 was reduced in the absence of phosphate. p31 was not associated with cyclin B, nor was it adsorbed to p13^suc1 Sepharose beads, two characteristics of p34^cdc2. p31 did, however, interact with p15, the starfish homologue of p13^suc1. p31 was present in cells in which cdc2⁺ was replaced by its budding yeast homologue CDC28. When fission yeast cytosolic extracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, the other at approximately 30 kDa. We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore, potentially, a new cdk. © 1997 John Wiley & Sons, Ltd.     

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

XV. Yeast sequencing reports. Sequence of a 10·27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene

A 10 270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size. The sequence has been deposited in the EMBL data library under Accession Number X75449.     

Secretion of mouse α-amylase from fission yeast Schizosaccharomyces pombe: Presence of chymostatin-sensitive protease activity in the culture medium

We have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse α-amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse α-amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse α-amylase than the 28 kDa killer secretion signal. We detected a chymostatin-sensitive protease activity in the culture medium of S. pombe, which digests mouse α-amylase secreted into the culture medium. The addition of 5 μg/ml chymostatin was shown to protect mouse α-amylases from this degradation.