(Verbatim from the applicant's description) In yeast and other fungi, cell shape, growth, and cell division depend on the coordinated synthesis and degradation of cell wall components. A quantitatively minor but functionally critical component of the yeast cell wall is the polymer beta-1,6 glucan. This polysaccharide is covalently cross-linked to other cell wall components, such as chitin, 3'-1,3 glucan and mannoproteins. These cross-links stabilize and organize the cell wall. The long-term goal of this project is to investigate the mechanisms by which beta-1,6 glucan is synthesized and associated with other cell wall components. We hypothesize that key aspects of beta-1,6 synthesis differ from those of the more abundant 3'-1,3 glucan polymer. Specifically, the proposed work will examine the hypothesis that beta-1,6 glucan synthesis is initiated in the lumen of the endoplasmic reticulum. Chain elongation and branching occurs in the Golgi apparatus, where the assembled polymers are packaged into secretory vesicles for delivery to the periplasmic space and assembly into the cell wall. In previous work we demonstrated the transport of UDP-glucose, the sugar donor for polymer assembly, into the lumen of the endoplasmic reticulum in Saccharomyces cerevisiae. In order to test our hypothesis we will identify the UDP-glucose transporter gene in yeast and generate a null mutant. Our model predicts that '3-1,6 glucan synthesis, but not 3'-1,3 glucan synthesis, will be prevented in the null mutant. A second, independent, strategy is to determine directly the subcellular sites in which beta-1,6 glucan chains are initiated and extended. This will be accomplished by in vivo radiolabeling experiments in combination with cell fractionation. This work will be facilitated by the use of mutants defective in beta-1,6 glucan synthesis (kre) and secretion (sec). The final approach consists of identifying and characterizing the enzymes that are responsible for synthesis of '3-1,6 glucan. The information derived from these studies will provide important new knowledge on the molecular mechanism of fungal cell wall assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059773-05
Application #
6752424
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Marino, Pamela
Project Start
2000-06-01
Project End
2006-05-31
Budget Start
2004-06-01
Budget End
2006-05-31
Support Year
5
Fiscal Year
2004
Total Cost
$209,170
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Uccelletti, Daniela; Farina, Francesca; Rufini, Silvia et al. (2006) The Kluyveromyces lactis alpha1,6-mannosyltransferase KlOch1p is required for cell-wall organization and proper functioning of the secretory pathway. FEMS Yeast Res 6:449-57
Krysan, Damian J; Ting, Elizabeth L; Abeijon, Claudia et al. (2005) Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae. Eukaryot Cell 4:1364-74
Farina, Francesca; Uccelletti, Daniela; Goffrini, Paola et al. (2004) Alterations of O-glycosylation, cell wall, and mitochondrial metabolism in Kluyveromyces lactis cells defective in KlPmr1p, the Golgi Ca(2+)-ATPase. Biochem Biophys Res Commun 318:1031-8
Herrero, Ana B; Magnelli, Paula; Mansour, Michael K et al. (2004) KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties. Eukaryot Cell 3:1423-32
Wheeler, Robert T; Kupiec, Martin; Magnelli, Paula et al. (2003) A Saccharomyces cerevisiae mutant with increased virulence. Proc Natl Acad Sci U S A 100:2766-70
Herrero, Ana B; Uccelletti, Daniela; Hirschberg, Carlos B et al. (2002) The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis, glycosylation, and cell wall properties. Eukaryot Cell 1:420-31
Magnelli, Paula; Cipollo, John F; Abeijon, Claudia (2002) A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure. Anal Biochem 301:136-50
Feoktistova, A; Magnelli, P; Abeijon, C et al. (2001) Coordination between fission yeast glucan formation and growth requires a sphingolipase activity. Genetics 158:1397-411