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Vol. 12, Issue 8, 2497-2518, August 2001
#
and¶
*Department of Biology and The bipolar budding pattern of a/Program in Molecular
Biology and Biotechnology, University of North Carolina, Chapel Hill,
North Carolina 27599; and Department of Biology, McGill
University, Montreal H3A 1B1, Canada
Saccharomyces
cerevisiae cells appears to depend on persistent spatial markers
in the cell cortex at the two poles of the cell. Previous analysis of
mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding
components of the markers at the poles distal and proximal to the birth
scar, respectively. Further genetic analysis reported here supports
this hypothesis. Mutants deleted for BUD8 or
BUD9 grow normally but bud exclusively from the proximal
and distal poles, respectively, and the double-mutant phenotype
suggests that the bipolar budding pathway has been totally disabled.
Moreover, overexpression of these genes can cause either an increased
bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position,
depending on the level of expression. The structures and localizations
of Bud8p and Bud9p are also consistent with their postulated roles as
cortical markers. Both proteins appear to be integral membrane proteins of the plasma 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
domains of the two proteins are very similar in sequence. When Bud8p
and Bud9p were 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 necks 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.
Surprisingly, Bud8p localization to the presumptive bud site and bud
tip also depends on actin but is independent of the septins.
Online version of this article contains video
material for Figure 9. Online version is available at
www.molbiolcell.org.
Present addresses:
#Swiss Institute
for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland;
§Botanisches Institut der Universität Basel, CH-4056
Basel, Switzerland;
Department of Biological Sciences,
Stanford University, Stanford, CA 94305.
¶
Corresponding author. E-mail:
jpringle{at}emailunc.edu.
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