Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1⌬ and eki1⌬ cki1⌬ mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1⌬ mutant, cki1⌬ mutant, and eki1⌬ cki1⌬ double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1⌬ mutants, and in cki1⌬ mutants indicated that ethanolamine was the preferred substrate. The eki1⌬ mutant did not exhibit a growth phenotype. Biochemical analyses of eki1⌬, cki1⌬, and eki1⌬ cki1⌬ mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1⌬ mutants, eki1⌬ mutants did not suppress the essential function of Sec14p.
Alkaline extracellular protease (AEP) from Yarmwia /ipo/'ica is synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Al a,, by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala-and -X-Pro-dipeptide stretch (Pro,, to Met which should prevent DPAPase processing and Al a,, to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor ProboroPro. Deletion of the entire dipeptide stretch (Ala,, to Pro, , ) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type and dpp1 dap2 Sacchammyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that the KEXZ-like cleavage after Lys, , , -Arg, , , , which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.
Soluble collagen, proteoglycans and two lysosomal enzymes known to degrade proteoglycans were studied during ice storage of skeletal muscle from Pacific rockfish. The solubility of muscle collagen progressively increased during storage in ice for 7 days. Proteoglycans isolated from prerigor muscle by extraction with guanidine HCl and density gradient ultracentrifugation contained only 43 μg hexuronic acid/100 g of wet tissue. The sulfated glycosaminoglycans portion of proteoglycans were isolated by trypsin digestion of an extract from muscle acetone powder and contained 58 μg hexuronic acid/20 g of acetone‐dried muscle tissue. Sulfated glycosaminoglycans had several components which separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Electrophoresis of sulfated glycosaminoglycans from stored rockfish revealed disappearance/decrease of specific zones after 5 days at 0C. The degradation of proteoglycans occurs in postmortem fish muscle and this may contribute to the destabilization of the extracellular matrix and texture softening during postharvest storage of fish.
The Yarrowia lipolytica SRP101 homologue encoding the signal recognition particle (SRP) receptor α‐subunit (SRαp) was cloned using degenerate primers designed for conserved GTP‐binding domains. Sequencing of 2814 nucleotides revealed an open reading frame of 1671 base pairs encoding a putative protein of 557 amino acids with a predicted molecular mass of 61 kDa. Like other SRP101 homologues, Y. lipolytica SRP101 contains a highly conserved C‐terminal GTP binding site. It has 44%, 34% and 22% sequence identity with S. cerevisiae, mammalian and Escherichia coli homologues, respectively. As found for SRP protein subunits of Y. lipolytica, SRP101 is important but not essential for cell growth. A conditional mutation in SRP101 affected synthesis/translocation of alkaline extracellular protease and Kar2p consistent with Srp101p functioning as an SRP receptor subunit. The SRP101 sequence has been deposited in GenBank under Accession No. AF132597. Copyright © 2000 John Wiley & Sons, Ltd.
In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (3 weeks journals - search completed 21st June 2000)
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