Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appea rs to depend on persistent spatial markers in the cell cortex at the two po les of the cell. Previous analysis of mutants with specific defects in bipo lar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectivel y. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proxim al and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, over expression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud positi on, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as corti cal markers. Both proteins appear to be integral membrane proteins of the p lasma membrane, and they have very similar overall structures, with long N- terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domai ns of the two proteins are very similar in sequence. When Bud8p and Bud9p w ere localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the n ecks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surpr isingly, Bud8p localization to the presumptive bud site and bud tip also de pends on actin but is independent of the septins.