Book of Abstracts (PDF) - International Mycological Association

IMC7 Monday August 12th Lectures
KRE6 encodes a glucanase-like protein required for beta-
1,6-glucan synthesis, and is synthetically lethal with the
related SKN1. KRE6 interacts synthetically with 87 genes,
and SKN1 with 39. Again there is little overlap between
the sets, with a total of 126 interactions among 121 genes.
These genes are mainly in areas of cell wall maintenance,
cell stress, protein degradation, protein modification, lipid
and fatty acid metabolism and vesicle transport. The
KRE6/SKN1 set show limited overlap with the FKS family
set, consistent with their distinct functions, with a total for
both families of 301 interactions among 286 different
genes.
33 - The PMTs: an evolutionarily conserved family of
protein O-mannosyltransferases
V. Girrbach, M. Lommel & S. Strahl *
University of Regensburg Department of Cell Biology and
Plant Physiology, Universitätsstrasse 31, D-93053
Regensburg, Germany. - E-mail: sabine.strahlbolsinger@biologie.uni-regensburg.de
Protein O-mannosylation is an essential protein
modification in yeast. Furthermore, it is indispensable for
cell morphology and cell wall integrity [1]. Yet, how Omannosylation
affects cell wall architecture is still obscure.
In yeasts and fungi O-mannosylation is initiated at the
endoplasmic reticulum by an evolutionarily conserved
family of protein O-mannosyltransferases, the PMTs.
Phylogenetic analyses revealed, that this family can be
divided into Pmt1p, Pmt2p and Pmt4p like subfamilies,
including transferases closely related to Pmt1p, Pmt2p and
Pmt4p, respectively. Here we present the molecular
characterization of the PMT subfamilies of S. cerevisiae
showing that Pmts are integral membrane proteins with
seven membrane spanning domains which form specific
high molecular weight complexes in vivo. Further,
characterizing pmt mutants we found that their phenotype
closely resembles mutants of the cell wall integrity
signaling (PKC1-) pathway which monitors cell wall
stability [2]. Analysis of the PKC1-pathway revealed that
O-mannosylation is essential for induction of the this signal
transduction cascade upon external stresses. Biochemical
analysis of the highly O-mannosylated upstream receptors
of the cell wall integrity pathway showed that abated Omannosylation
affects their maturation, stability and
function. References [1] Strahl-Bolsinger S et al. (1999)
Biochim. Biophys. Acta 1426: 297-307 [2] Heinisch JJ et
al. (1999) Mol Microbiol 32: 671-680.
34 - Regulation of glycosylation and pH in the yeast
Golgi
N. Dean * & X.D. Gao
Department of Biochemistry and Cell Biology, Institute for
Cell and Developmental Biology, State University of New
York, Stony Brook, NY 11794-5215, U.S.A. - E-mail:
Neta.Dean@stonybrook.edu
In yeast, glycoproteins are heavily modified by mannose in
the Golgi. The donor for mannosylation is GDP-mannose.
Use by lumenal mannosyltransferase requires that GDPmannose
be transported from the cytosol to the Golgi by a
specific transporter. Once the mannose is donated to
protein, GDP is converted to GMP by nucleoside
diphosphatases. GMP is an antiporter whose export from
the Golgi is coupled to the lumenal import of GDPmannose.
Substrate provision thus involves a cycle in
which mannosylation acts as a sink to continuously
generate the antiporter. As a byproduct of this cycle,
hydrolysis of GDP to GMP generates a huge amount of
phosphate that must be removed to prevent an overly acidic
lumenal pH. Existence of a phosphate transporter is
hypothesized to be critical for pH homeostasis in the Golgi,
as mannosylation in the Golgi accounts for the vast
majority of cellular GDP hydrolysis. We present evidence
that ERD1 plays a key role in regulating lumenal Golgi pH.
Erd1p is Golgi localized and is homologous to other
phosphate transporters. erd1 mutants suffer from a number
of Golgi defects, including glycosylation and ER protein
retention. erd1 mutants are EGTA sensitive,show pH- and
PO4-dependent growth defects and are suppressed by
ERS1, a gene that functions to regulate vacuolar pH. Taken
together our results support the model that Erd1p regulates
the removal of lumenal phosphate that is generated through
the consumption of nucleotide sugars during glycosylation
in the Golgi.
35 - Environmental stimuli suppress the Neurospora
crassa cot-1 phenotype
R. Gorovits * & O. Yarden
Hebrew University of Jerusalem, Faculty of Agriculture,
Rehovot 76100, Israel. - E-mail: gorovits@agri3.huji.ac.il
TheNeurospora crassa colonial temperature sensitive-1
(cot-1) gene encodes a Ser/Thr protein kinase required for
proper hyphal elongation. The temperature-sensitive cot-1
strain exhibits normal spreading radial growth at or below
25 °C, but mutant colonies grow slowly with extensively
branched hyphae at or above 32 °C. Antibodies raised
against COT1 detect a 67-kDa polypeptide that is absent in
extracts obtained from cot-1 grown at restrictive
conditions. The cot-1 hyperbranching phenotype is
accompanied by an increase in proton efflux (as
determined by rate of medium acidification) and a 50-75%
reduction in relative intracellular sodium content
(determined by X-ray microanalysis). Ammending the
growth medium with ion pump inhibitors (DES, Amiloride
or Ouabain), NaCl (0.5-1.5M) or sorbitol (1-1.5M)
remedies the ionic imbalance and suppresses the cot-1
phenotype to various degrees. The COT1 67kDa
polypeptide was detected in extragenic suppressors of cot-1
(exhibiting partial or full suppression), but not in cot-1
cultures suppressed by the tested environmental stimuli.
Based on these results we suggest that impaired COT1
function confers changes in cellular ionic homeostasis and
that suppression of the cot-1 phenotype by genetic
alterations versus changes in environmental conditions may
involve different pathways.
Book of Abstracts 13