The yeast SEC53 gene encodes phosphomannomutase

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Yeast sec53 mutants accumulate incompletely glycosylated secretory proteins in the endoplasmic reticulum.
• A defect in alpha-factor precursor glycosylation was observed in vitro using sec53 mutant cell components.

Purpose of the Study:

• To investigate the molecular basis of the glycosylation defect in yeast sec53 cells.
• To identify the gene product responsible for the observed phosphomannomutase deficiency.

Main Methods:

• In vitro glycosylation assays using isolated membranes and cytosol.
• Enzyme activity assays for phosphomannomutase and phosphomannoisomerase.
• Complementation studies with wild-type fractions, GDP-mannose, and mannose phosphates.
• Gene complementation and protein expression analysis.
• Protein purification and cofractionation studies.

Main Results:

• sec53 cell extracts showed deficient phosphomannomutase activity but normal phosphomannoisomerase activity.
• Supplementation with GDP-mannose or mannose 1-phosphate restored normal glycosylation.
• SEC53 gene product was identified as yeast phosphomannomutase.
• The Sec53 protein and phosphomannomutase activity cofractionated during purification.

Conclusions:

• The SEC53 gene encodes yeast phosphomannomutase, an enzyme crucial for GDP-mannose synthesis.
• Deficiency in phosphomannomutase activity leads to a deficit in GDP-mannose production.
• This deficit explains the secretory protein glycosylation defect observed in sec53 cells.