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The isolation of a Dol-P-Man synthase from Ustilago maydis that functions in Saccharomyces cerevisiae
J W Zimmerman1, C A Specht, B X Cazares
1Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
Abstract:
Genomic DNAs from several fungi were screened for a homologous sequence to Saccharomyces cerevisiae DPM1, an essential gene which encodes dolichyl phosphoryl mannose synthase. The fungi examined included Aspergillus nidulans, Neurospora crassa, Schizophyllum commune and Ustilago maydis. Only U. maydis gave a significant signal after Southern hybridization using DPM1 as a probe. The Ustilago homolog was subsequently cloned and sequenced. The predicted protein of 294 amino acids has 60% identity to the S. cerevisiae protein, but lacks the putative "dolichol recognition sequence'. RNA of ca. 900 bp is transcribed in both yeast and filamentous cells of Ustilago. In Escherichia coli, the U. maydis sequence expressed a 35 kDa protein exhibiting dolichyl phosphoryl mannose synthase activity. The sequence was also shown to complement a haploid strain of S. cerevisiae containing a deletion of the DPM1 gene. The U. maydis sequence therefore, encodes a dolichyl phosphoryl mannose synthase that can support normal vegetative growth in S. cerevisiae.
Insights
Researchers identified a homologous gene in Ustilago maydis that encodes dolichyl phosphoryl mannose synthase. This essential enzyme supports normal growth in yeast, demonstrating functional conservation across fungal species.
Area of Science:
- Molecular Biology
- Mycology
- Biochemistry
Background:
- The Saccharomyces cerevisiae DPM1 gene is essential for dolichyl phosphoryl mannose synthesis.
- Dolichyl phosphoryl mannose is crucial for N-linked glycosylation in eukaryotes.
Purpose of the Study:
- To identify and characterize fungal homologs of the essential Saccharomyces cerevisiae DPM1 gene.
- To investigate the functional conservation of dolichyl phosphoryl mannose synthase across different fungal species.
Main Methods:
- Southern hybridization to screen fungal genomic DNAs.
- Cloning and sequencing of the Ustilago maydis DPM1 homolog.
- Functional expression in Escherichia coli and complementation of a yeast DPM1 deletion mutant.
Main Results:
- A homologous sequence to S. cerevisiae DPM1 was identified only in Ustilago maydis.
- The U. maydis homolog encodes a 294-amino acid protein with 60% identity to S. cerevisiae DPM1.
- The U. maydis protein exhibits dolichyl phosphoryl mannose synthase activity and complements a yeast DPM1 deletion, supporting normal growth.
Conclusions:
- Ustilago maydis possesses an essential gene encoding dolichyl phosphoryl mannose synthase.
- The U. maydis dolichyl phosphoryl mannose synthase is functionally conserved and can support essential cellular processes in Saccharomyces cerevisiae.