ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

Ralf-Peter Jansen
Asymmetric Cell Division and RNA
Transport in Yeast
During embryogenesis, cell type diversity is generated
by different mechanisms, one of which is asymmetric
cell division. Such divisions produce progeny differing
in morphology, size, or gene expression pattern.
However, asymmetric divisions are not only found in
multicellular organisms. A typical unicellular eukaryote
that divides asymmetrically is the yeast Saccharomyces
cerevisiae. A „mother cell“ generates a
„daughter cell“ by polarized growth, a process known
as „budding“. After separation, the two cells show
a differential gene expression pattern. Whereas the
mother cell can express the HO gene and eventually
undergoes a process called „mating type switching“,
the daughter cell is unable to do so.
HO expression depends on multiple positively and
negatively acting transcription factors but the differential
expression pattern of HO is regulated by the
asymmetric distribution of a transcriptional repressor
known as Ash1p. Ash1p can be detected in post-anaphase
cells predominantly in the nucleus of daughter
cells where it persists to the end of the daughter cell‘s
following G1 phase. In the daughter cell it is both sufficient
and essential to shut off HO expression in G1.
The asymmetric distribution of Ash1p is the result of
an asymmetric localization of its mRNA during anaphase.
ASH1 mRNA is transported from the nuclei of
both mother and daughter cell to the cell cortex of
the daughter. The localization depends on a functional
microfilament system and three cis-acting localization
signals. These signals appear to be independent of each
other since each signal is sufficient to target a reporter
RNA to the daughter cell. In addition to the cis signals,
5 so called She proteins are essential for ASH1 mRNA
localization. Among these proteins is a type V myosin
80
(She1p/Myo4p) and She5p/Bni1p, a member of the
FH („formin homology“) protein family required for
actin cytoskeleton organization in different organisms.
Homologs of another She protein, She4p have recently
been predicted to be involved in myosin assembly or
function. The last two She proteins, She2p and She3p
do not show any sufficient homology to other proteins.
Myo4p, a motor protein essential for mRNA
transport
D. Djandji, A. Frank, C. Kruse, S. Münchow, and C.
Sauter
To date, Myo4p is the only motor protein with an essential
role in mRNA localization. In order to understand
its role we initially wanted to test if the myosin
is directly involved in ASH1 mRNA transport.
Using a combination of in-situ hybridization and indirect
immunofluorescence, we could show a colocalization
of the myosin and ASH1 mRNA not only at the
final targeting site (the tip of the daughter cell) but
also on filamentous structures that are most likely microfilaments
running from the mother to the daughter
cell (Fig. 1). In addition, ASH1 mRNA was shown to
specifically coprecipitate with an epitope-tagged version
of Myo4p. Both sets of data strongly suggest that
ASH1 mRNA is in fact a cargo of the Myo4p myosin.
What other proteins are required for the association of
ASH1 mRNA with Myo4p? One candidate was She3p
since it‘s intracellular localization was very similar
to that of Myo4p. We were able to demonstrate that
She3p is essential for Myo4p-ASH1 association. Furthermore,
She3p coprecipitated both with Myo4p and
Figure 1
ASH1 mRNA. In addition, She3p‘s aminoterminus
that is predicted to form a coiled coil can bind to
the carboxyterminal tail of Myo4p in a two hybrid assay
whereas She3p‘s carboxyterminus interacts with
She2p. She3p and She2p might be adapters between
the mRNA and the motor protein.
The role of Myo4p‘s tail domain was further investigated
since the tail domains of other myosins are
involved in cargo binding. Expression of the tail in an
otherwise wildtype cell had the effect of a dominant
negative mutant. It abolished the localization of ASH1
mRNA, She3p and endogenous Myo4p as well as the
association of endogenous Myo4p with She3p, most
likely by sequestering essential factors. The effects are
specific since the expression of another type V myosin
tail did not interfere with mRNA localization.
The myosin tail domain should be useful to biochemically
identify and copurify components of the putative
mRNA transport complex, especially the RNAbinding
factor(s).
Ash1p as a regulator of pseudohyphal differentiation
K. Kahlina and S. Münchow
Besides it‘s role in regulating HO expression and
mating type switching, Ash1p is required for another
differentiation process in Saccharomyces cerevisiae,
pseudohyphal growth. During this differentiation,
yeast cells elongate and grow unipolar to form chains
of connected cells that allow a yeast colony to penetrate
it‘s medium. Several signalling pathways converge
during induction of pseudohyphal growth and
at least four transcription factors (including Ash1p)
are required for this differentiation process. However,
nothing is known about the signalling pathway that
regulates Ash1p or about the target genes that are regulated
by Ash1p.
We have recently shown that mislocalization of ASH1
mRNA and Ash1 protein in a myo4 mutant dramatically
increases pseudohyphal growth suggesting that
RNA targeting plays a crucial role in proper pseudohyphal
differentiation. In order to identify ASH1-dependent
genes, two independent screens based on dif-
81