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Vol. 20, Issue 22, 4856-4870, November 15, 2009

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Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

Eleonora Rolli^*,,, Enrico Ragni^*,, Julia Calderon^*, Silvia Porello, Umberto Fascio^||, and Laura Popolo^*

*Dipartimento di Scienze Biomolecolari e Biotecnologie and ^||Centro Interdipartimentale Microscopia Avanzata, Università degli Studi di Milano, 20133 Milano, Italy; and Department of Chemistry, Ursinus College, Collegeville, PA 19426

Submitted December 1, 2008; Revised September 8, 2009; Accepted September 17, 2009
Monitoring Editor: Howard Riezman

Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3 cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2 mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope.

These authors equally contributed to this work.

Present address: Dipartimento di Scienze e Tecnologie Agroalimentari e Microbiologiche (DiSTAM) Università degli Studi di Milano, 20133 Milano, Italy.

Address correspondence to: Laura Popolo (laura.popolo{at}unimi.it)

Abbreviations used: -F, -factor; CF, calcofluor white.