ROLE OF BUD3P IN PRODUCING THE AXIAL BUDDING PATTERN OF YEAST

Yeast cells can select bud sites in either of two distinct spatial pat terns. a cells and alpha cells typically bud in an axial pattern, in w hich both mother and daughter cells form new buds adjacent to the prec eding division site, In contrast, a/alpha cells typically bud in a bip olar pattern, in which new buds can form at either pole of the cell. T he BUD3 gene is specifically required for the axial pattern of budding : mutations of BUD3 (including a deletion) affect the axial pattern bu t not the bipolar pattern. The sequence of BUD3 predicts a product (Bu d3p) of 1635 amino acids with no strong or instructive similarities to previously known proteins. However, immunofluorescence localization o f Bud3p has revealed that it assembles in an apparent double ring enci rcling the mother-bud neck shortly after the mitotic spindle forms. Th e Bud3p structure at the neck persists until cytokinesis, when it spli ts to yield a single ring of Bud3p marking the division site on each o f the two progeny cells, These single rings remain for much of the ens uing unbudded phase and then disassemble. The Bud3p rings are indistin guishable from those of the neck filament-associated proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p), except that the latter proteins assemble before bud emergence and remain in place for the duration of the cell cycle. Upon shift of a temperature-sensitive cdc12 mutant to restricti ve temperature, localization of both Bud3p and the neck filament-assoc iated proteins is rapidly lost, In addition, a haploid cdc11 mutant lo ses its axial-budding pattern upon shift to restrictive temperature. T aken together, the data suggest that Bud3p and the neck filaments are linked in a cycle in which each controls the position of the other's a ssembly: Bud3p assembles onto the neck filaments in one cell cycle to mark the site for axial budding (including assembly of the new ring of neck filaments) in the next cell cycle. As the expression and localiz ation of Bud3p are similar in a, alpha, and a/alpha cells, additional regulation must exist such that Bud3p restricts the position of bud fo rmation in a and alpha cells but not in a/alpha cells.