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vga Mutants of Kluyveromyces lactis show cell integrity defects
D Uccelletti1, V Pacelli, P Mancini
1Fondation Institut Pasteur-Fondazione Cenci-Bolognetti, Department of Developmental and Cell Biology, University of Rome La Sapienza, Rome, Italy.
Abstract:
We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.
Insights
Mutants of Kluyveromyces lactis with impaired glycosylation exhibit altered cell walls. These vanadate glycosylation affected (vga) mutants show changes in beta-D-glucan and chitin, impacting cell wall structure and integrity.
Area of Science:
- * Mycology
- * Cell Biology
- * Biochemistry
Background:
- * Glycosylation is crucial for fungal cell wall synthesis and integrity.
- * Kluyveromyces lactis glycosylation mutants (vga1-vga4) were previously identified.
- * These mutants display resistance to sodium orthovanadate and altered invertase glycosylation.
Purpose of the Study:
- * To investigate cell wall alterations in Kluyveromyces lactis glycosylation mutants.
- * To characterize the impact of impaired glycosylation on cell wall composition and structure.
- * To explore the relationship between glycosylation and beta-D-glucan synthesis.
Main Methods:
- * Analysis of cell wall composition (beta-D-glucan, chitin) in wild-type and mutant strains.
- * Detection and quantification of Fks1p-like protein using Western blotting.
- * Electron microscopy for visualizing cell wall ultrastructure.
- * Zymolyase sensitivity assays to assess cell wall integrity.
Main Results:
- * vga1, vga2, and vga3 mutants showed reduced alkali-soluble beta-D-glucan and increased chitin.
- * vga4 mutants had reduced levels of both alkali-soluble and alkali-insoluble beta-D-glucan with normal chitin.
- * Fks1p-like protein levels increased 7-10 fold in vga1, vga2, and vga3 mutants.
- * Mutants exhibited thicker cell walls with structural irregularities and increased beta-D-glucan release.
Conclusions:
- * Impaired glycosylation significantly affects Kluyveromyces lactis cell wall composition and structure.
- * Mutations impacting glycosylation lead to altered beta-D-glucan synthesis and accumulation.
- * Kluyveromyces lactis serves as a valuable model for studying glycosylation-cell wall interconnections.