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PMI40, an intron-containing gene required for early steps in yeast mannosylation
D J Smith1, A Proudfoot, L Friedli
1Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
Abstract:
We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.
Insights
Researchers isolated the PMI40 gene in Saccharomyces cerevisiae, crucial for phosphomannose isomerase (PMI) activity. Deleting this gene necessitates D-mannose for yeast growth, revealing new regulatory insights.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- A temperature-sensitive mutant (pmi40-1) in Saccharomyces cerevisiae exhibits defects in glycosylation and secretion due to thermolabile phosphomannose isomerase (PMI) activity.
- Inactivation of PMI at 37°C halts the synthesis of essential mannosyl donors, leading to cell death.
Purpose of the Study:
- To isolate the PMI40 gene responsible for phosphomannose isomerase activity in Saccharomyces cerevisiae.
- To characterize the structure and function of the PMI40 gene and its encoded protein.
- To investigate the regulation of PMI40 gene expression.
Main Methods:
- Complementation of the pmi40-1 mutation to isolate the PMI40 gene.
- Analysis of the PMI40 gene's intron structure.
- Protein prediction and comparison with purified PMI peptides.
- Gene deletion to assess the requirement for D-mannose.
- Quantitative analysis of PMI40 gene transcription under different carbon sources.
Main Results:
- The PMI40 gene was successfully isolated by complementation.
- PMI40 contains a unique, short intron with an AACTAAC branch motif.
- Deletion of PMI40 renders yeast auxotrophic for D-mannose.
- PMI40 transcription increases 12-fold on D-mannose, but PMI enzyme activity and protein levels remain constant, indicating post-transcriptional regulation.
Conclusions:
- The PMI40 gene encodes the essential phosphomannose isomerase in Saccharomyces cerevisiae.
- The PMI40 gene exhibits unusual intron splicing and is subject to complex regulatory mechanisms.
- Understanding PMI40 regulation provides insights into yeast mannose metabolism and glycosylation pathways.