ABSTRACT
In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.
Subject(s)
Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/physiology , Genes, Essential , Microbial Viability/genetics , Pichia/genetics , Pichia/physiology , Fungal Proteins/genetics , Fungal Proteins/physiology , Gene Deletion , Glycosylation , Mutagenesis, Insertional , Pichia/growth & development , Protein Transport/genetics , Recombinant Proteins/metabolism , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP-mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP-mannose, such as cell wall fragility and defects in N- and O-glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum-associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177.
Subject(s)
Nucleotidyltransferases/genetics , Pichia/genetics , Amino Acid Sequence , Base Sequence , Cell Wall/metabolism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/metabolism , Glycosylation , Microscopy, Electron , Molecular Sequence Data , Mutation , Nucleotidyltransferases/physiology , Pichia/enzymology , Pichia/physiology , Urokinase-Type Plasminogen Activator/biosynthesis , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
Using a screening procedure for obtaining yeast strains with enhanced ability to secrete heterologous protein, we have isolated a mutant with alteration of the cell wall structure. This mutant displayed strong decrease in cell wall mannoprotein content, which was not accompanied by decreased glycosylation of secreted proteins. The mutation defines a gene, designated SSU21(identical to previously characterized MCD4), which encodes a novel vacuolar protein. SSU21 is probably connected to the cell integrity protein kinase C-mediated pathway, since ssu21 and pkc1Delta double mutant is synthetic lethal. To our knowledge, this is the first example of a yeast vacuolar protein whose alteration results in a cell wall defect.
