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Sln1: All species investigated have at least one, in many instances several, histidine
kinases (see also (Catlett et al. 2003)). To classify as an Sln1 orthologue, the protein had
to possess two predicted N-terminal transmembrane domains in addition to the histidine
kinase and response regulator domain. Each genome investigated encoded one such
protein, with the exceptions of S. pombe and U. maydis, which both have three different
histidine kinases lacking transmembrane domains. The sequences from S. kluyveri and D.
hansenii were significantly shorter due to frameshifts, presumably resulting from
sequencing errors. For S. kluyveri, the DNA sequence could be used to restore the likely
open reading frame. Sequence alignments and domain structure of Sln1 and the other two
phosphorelay proteins are discussed in the accompanying paper (Krantz et al. 2005b).
Ypd1: All genomes investigated encoded one single phosphotransfer protein. These are
small proteins of 15620 residues, excluding Sz. pombe Spy1, which is significantly
larger due to an N-terminal extension (295aa, p=2.9*10-6). It had already been noted
(Catlett et al. 2003) that apparently one phosphotransfer protein is sufficient even if an
organism has multiple histidine kinases.
Ssk1: Ssk1 is characterised by a response regulator domain. One putative orthologue was
identified per species, with the exception of U. maydis, which seems to possess two.
However, the second candidate from U. maydis, UM05594.1, has a response regulator
domain that does not cluster with the Ssk1 orthologues and hence probably has a different
function (Krantz et al. 2005b).
Ssk2/Ssk22: These are large proteins of 155473 and 137850 amino acids, respectively,
excluding the much larger protein from U. maydis. They consist of an N-terminal
regulatory and C-terminal protein kinase catalytic domain. Excluding the most proximal
part, Ssk2 and Ssk22 are 53% identical. Ssk2 and Ssk22 appear to have redundant
function in the S. cerevisiae HOG pathway, although Ssk2 has been proposed to have a
separate role in actin cytoskeleton recovery (Yuzyuk et al. 2002). Outside the
Saccharomyces genus, only Sz. pombe has two similar MAPKKKs, while in all other
species we only found a single putative Ssk2/Ssk22 orthologue. Ssk2/Ssk22 from
different organisms only align well within the kinase domain. In contrast, size is rather
well conserved, with a coefficient of variation of 4.7 and 3.6%, respectively. The
orthologue from A. nidulans appears to be missanotated, as the sequence contains both
the MAPKKK domain and a fungal transporter domain. The sequence corresponding to
the other filamentous ascomycetes is included as a restored ORF but excluded from
further analysis.
Pbs2: A likely candidate for a Pbs2 was found in each species, with the exception of F.
graminearum and N. crassa, which both have two. These additional copies of Pbs2-like
proteins are quite similar to each other (73% identity) but only have limited similarity to
Pbs2 from all other fungal species (less than 30% identity). The predicted U. maydis Pbs2
is significantly shorter (p=4*10-4), lacks the variable N-terminal domain and only
contains the better conserved kinase domain. Pbs2 is discussed in more detail in the
accompanying paper (Krantz et al. 2005b).
Hog1: Each species has one clearly identifiable Hog1 orthologue and all are more than
70% identical to S. cerevisiae Hog1. The A. nidulans genome encodes one potential
additional Hog1 orthologue (called MpkC), which, however, is only 62% identical to S.
cerevisiae Hog1 and possibly has a different function (Furukawa et al. 2005). The Hog1
orthologues from yeasts are significantly longer than their filamentous counterparts
(42031 instead of 36410, p=7.1*10-6, t-test).
Ptp2 and Ptp3: These two phosphotyrosine protein phosphatases are only 23% identical
to each other and similarity is mainly focused on the C-terminal catalytic domain. Ptp2
and Ptp3 have partially redundant roles in S. cerevisiae as negative regulators of Hog1,
but seem to perform their function in different compartments: Ptp2 in the nucleus and
Ptp3 in the cytosol (Mattison and Ota 2000; Saito and Tatebayashi 2004). Indeed, species
more closely related to S. cerevisiae, incl. A. gossypii and K. waltii, seem to have two
such proteins. The syntenic orthologues in A. gossypii are 34 and 35% identical for Ptp2
and Ptp3, respectively. Hence, orthologues are more similar to each other than Ptp2 and
Ptp3 in S. cerevisiae. In more distantly related species identification becomes more
doubtful with identity scores around, or less than, 20%. BLAST searches starting from
Ptps from filamentous fungi commonly identify S. cerevisiae Ptp1, which is not a
component of the HOG pathway (Saito and Tatebayashi 2004). Furthermore, they seem
to be larger than in yeasts and the putative Ptp3 from U. maydis is predicted to be
particularly large (1683 amino acids).
Ptc1 and Ptc2/Ptc3: These three phosphothreonine phosphatases seem to have partly
overlapping functions in S. cerevisiae. It appears that Ptc1 plays the most prominent role
in the HOG pathway and, like Ptc2 and Ptc3, has additional roles in cell physiology
(Saito and Tatebayashi 2004). While Ptc2 and Ptc3 are highly similar to each other (60%
identity), they are clearly different from Ptc1 (24 and 29% identity, respectively), and a
forth protein, Ptc4 (25 and 26% identity, respectively). They also has a smaller Cterminal domain as compared to the other Ptc proteins; this domain is significantly larger
in filamentous fungi, which is reflected in the protein size (53995 instead of 30039;
p=3.9*10-3, t-test). In contrast to the Saccharomyces, others yeasts only seem to have
Ptc1 and Ptc3 orthologues. A. gossypii has a syntenic homologue to Ptc1 and Ptc3 but not
to Ptc2. Similarly, filamentous fungi seem to have only two Ptc proteins, where one is
likely a Ptc1 orthologue, while the second one resembles Ptc2 or Ptc3.
Sho1: This is a particularly interesting protein because it seems to play a role in several
MAPK pathways as a scaffold protein (O'Rourke and Herskowitz 1998; Raitt et al. 2000;
Seet and Pawson 2004; Zarrinpar et al. 2004). It is discussed in further detail in the
accompanying paper (Krantz et al. 2005b). We could identify likely orthologues in all
species except Sz. pombe, based on two criteria: N-terminal transmembrane domains
(based on experimental data on S. cerevisiae any membrane anchoring suffices for
function (Raitt et al. 2000)) and a C-terminal SH3 (Src homology 3) domain. The Sho1like proteins found in filamentous fungi were significantly smaller than the yeast
counterparts (p=0.0016, t-test).
Cdc42: This 191 amino acid essential Rho-like G-protein is conserved from yeast to
human and the best conserved protein studied here. Orthologues were more than 70%
identical in all species studied, except in Y. lipolytica whose Cdc42 is only 63% identical
to that of S. cerevisiae. The five filamentous fungi have an additional Cdc42-like protein.
Although these second Cdc42 homologues are highly similar to S. cerevisiae Cdc42 (5465% identity), they cluster together with each other and the orthologue of Y. lipolytica
(71-93% identity). They are of similar size, with the exception of the significantly smaller
protein from N. crassa (p=4.5*10-4). Likewise, K. waltii has an additional copy that is
distinct from either group, as well as significantly larger (p=9.2*10-24).
Cdc24: Likely orthologues of the Cdc42 GTP/GDP exchange factor (GEF) were found in
all species. Sequence identity, however, drops to about 20% for filamentous fungi. There
is a significant size difference between yeasts, 81646, and filamentous fungi, 101992
(p=0.0059, t-test).
Ste50: This Ste20-Ste11 adaptor protein is required for Ste11 activation (Ramezani-Rad
2003). Its exact biochemical role is poorly understood. There is one candidate orthologue
in each species, though sequence identity drops to about 20% in filamentous fungi. Size
in yeasts, 35253, and filamentous ascomycetes, 4886, is significantly different
(p=5.7*10-8, t-test). The U. maydis orthologue is significantly larger (829 residues,
p=1.3*10-5).
Ste20 and Cla4: Ste20 and Cla4 are PAKs, or p21-activated kinases. There is a third such
protein (Smk1) encoded by the S. cerevisiae genome, which seems to function in the
spore morphogenesis pathway. While Ste20 and Cla4 are only moderately similar (43%
identity in the kinase domain) each of them shows a higher degree of similarity to its
predicted orthologues. It appears that Cla4 takes part in Sho-branch signalling together
with Ste20 but analysis is hampered by the fact that a ste20 cla4 double mutant is
inviable (Raitt et al. 2000). It appears that almost all species studied here have both a
Ste20 and a Cla4 orthologue, with the exception of M. grisea and F. graminearum where
we could not identify a Ste20 orthologue, and C. glabrata, which seems to have an
additional Ste20 sequence. Cla4 is significantly, and Ste20 much, smaller in Sz. pombe
(p=2.3*10-4 and p=0.0069, respectively).
Ste11: All organisms studied here have one apparent Ste11 MAPKKK orthologue.
Overall sequence identity drops to just over 30% in the filamentous fungi. It is
considerably larger in U. maydis, 1568 amino acids as compared to 829112 in the
filamentous ascomycetes. Yeast Ste11 orthologues are 73147 amino acids, excluding
the significantly larger Y. lipolytica protein (944 residues, p=6.8*10-4). The Ste11 from S.
bayanus is only 437 residues; the DNA sequence corresponding to the first half of the S.
cerevisiae gene is missing.
Hot1 and Msn1: These two helix-loop-helix proteins belong to a small family of four
yeast transcription factors, which also includes Gcr1 and Ymr111. The sequence
similarity of Hot1 and Msn1 is restricted to the predicted DNA-binding domain (43%
identity) but there is evidence that both proteins have overlapping roles in HOGdependent gene expression (Rep et al. 1999; Alepuz et al. 2003). In most yeasts,
including A. gossypii and K. waltii, we found putative orthologues for both proteins. Due
to poor sequence conservation identification in filamentous fungi and more distantly
related yeasts is doubtful. Hot1 is discussed in more detail in the accompanying paper
(Krantz et al. 2005b).
Msn2/Msn4: These two general stress response factors are only about 27% identical but
the C-terminal 60 amino acids, which encompass the DNA-binding domain, are 78%
identical. There function is likely redundant. While Saccharomyces species seem to have
both an Msn2 and an Msn4 orthologue, other yeasts such as A. gossypii and K. waltii only
have one. In more distant yeasts and in filamentous fungi detection of candidate
orthologues is doubtful.
Sko1: This CRE-binding protein mediates Hog1-dependent transcription and it appears
that there is one candidate orthologue in other yeasts, with the exception of Sz. pombe,
which contains three candidate orthologues. Although their size is comparable to that of
the other Sko1 orthologues, the sequences are not conserved outside the DNA binding
domain. Due to low sequence conservation identification is doubtful in filamentous fungi.
Sko1 will be discussed in more detail in the accompanying paper (Krantz et al. 2005b).
Smp1: This transcriptional regulator appears to mediate a subset of Hog1-dependent
transcriptional responses (de Nadal et al. 2003), but does not seem to be the only factor
for any of the genes studied. Likely orthologues could be found in each organism except
K. waltii and M. grisea. The orthologues from filamentous fungi are significantly longer
than their yeast counterparts (p=0.0027, t-test).
Rck1/Rck2: These are two related (47% overall identity) MAPKAP (MAPK-activated
kinases) that mediate Hog1-dependent responses such as altered translational activity and
oxidative stress tolerance (Teige et al. 2001; Bilsland et al. 2004). Only the
Saccharomyces sensu strictu yeasts have both Rck1 and Rck2 orthologues. All other
putative orthologues identify Rck2 when used in BLAST searches. Rck2 is apparently the
more important paralogue in S. cerevisiae. While this may suggest that the role of Rck1
and Rck2 is largely redundant, the protein is duplicated in four additional yeasts (Table
4).
Msb1: Msb1 is a protein of unknown molecular function that genetically interacts with
Cdc42. A role in HOG signalling has not been demonstrated. Although we found
potential orthologues in all organisms except U. maydis, low sequence conservation
makes identification doubtful beyond yeasts closely related to S. cerevisiae and A.
gossypii. The orthologues in Y. lipolytica and K. waltii are significantly different in size
(p=4.9*10-5 and p=9.3*10-4, respectively). Yeast Msb1 orthologues are significantly
longer than those from filamentous fungi (p=0.0025, t-test).
Msb2: This is a mucin-like protein with one transmembrane domain that has recently
been reported to function upstream of Cdc42-dependent signalling in the HOG and the
filamentous growth pathway (Cullen et al. 2004). There are numerous onetransmembrane spanners in yeast (e.g. the Wsc proteins) that might play a role in
connecting the cell wall with the plasma membrane and intracellular components, such as
the actin cytoskeleton. We found potential orthologues in most species but low sequence
conservation makes identification in filamentous fungi doubtful. Size variance is large,
with a coefficient of variation of 19% in yeast and 18% in filamentous fungi.
Rtn1/Rtn2: RTN stands for Reticulon, in this case reticulon-like A subfamily. These
proteins have two transmembrane domains and are overall 28% identical. The Rtn1
candidates in Y. lipolytica and C. albicans are significantly larger (p=2.4*10-6 and 8.3*106
, respectively). This is also the case for the Rtn2 candidate in S. paradoxus, which has
been annotated from a different start twenty-four residues upstream of the perfectly
conserved start of all other Saccharomyces species. In filamentous fungi, which only
have homologues of Rtn1, they tend to be larger but the variation in size is greater
(35650 vs. 2939). There is evidence that Rtn2 interacts with Sho1 (Ito et al. 2001).
Rtn2 is only found in the Saccharomyces sensu strictu group, which explains their
extremely low coefficient of variation (0.2%). Still, Rtn1 has a coefficient of variation of
only 3,2%, less than half that of Hog1, 6.9%, in the same set species.
Bem1: Bem1 functions as a scaffold protein for complexes that include, among others,
Cdc42, Ste20 and Rsr1 (see below). A specific role in the Sho1 branch of the HOG
pathway has, however, so far not been demonstrated. Bem1 has two SH3 domains, one
PX, or Phox homology, domain and a C-terminal PB1, or Phox and Bem1, domain. We
found one apparent orthologue in each species investigated. Sequence identity drops to
about 30% in filamentous fungi.
Bem4: Also Bem4 is involved in protein complexes together with Cdc42 and affects cell
polarity and morphology, but a role in HOG pathway signalling has not been
demonstrated. The sequence is too poorly conserved to identify orthologues in distantly
related yeasts or filamentous fungi, as it is down to 32% identity already between S.
cerevisiae and A. gossypii. Size, however, is relatively well conserved with a coefficient
of variation of 3.7%, which is comparable to Sho1 (3.8%).
Bud6: Also Bud6 interacts with proteins like Cdc42, Cla4, Ste11 and Gic2 (see below) as
well as actin and it functions in establishing cell polarity, but a role in HOG signalling
has not been demonstrated. The protein has an AIP3 (actin-interacting protein 3) domain.
Likely orthologues were found in all species except for M. grisea and identity drops to
23% in U. maydis. The orthologue in Sz. pombe is significantly longer than those of other
yeasts (p=5.4*10-8), as are those from filamentous fungi (p=0.0047, t-test).
Far1: Far1 is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest and this
role is best studied in response to pheromone. Also Far1 interacts with Cdc42 and Cdc24.
The protein is only poorly conserved and overall sequence identity drops below 20%
already in distantly related yeasts (20% between S. cerevisiae and A. gossypii). However,
size in filamentous ascomycetes is well conserved, with a coefficient of variation of only
0.29%. Those putative Far1 orthologues are significantly longer than the yeast
counterparts (11623 and 863116, respectively; p=2.1*10-6, t-test), which show a higher
size variation.
Fus1: The protein is proposed to coordinate signalling, fusion, and polarisation events
required for fusion. Recently it has been reported that Fus1 interacts with the SH3
domain of Sho1 and interaction with Cdc42 and Ste50 has also been reported (Nelson et
al. 2004). Fus1 has an SH3 domain itself as well as a transmembrane domain. Overall
sequence identity, however, drops below 20% already in distantly related yeasts (22%
identity between S. cerevisiae and A. gossypii Fus1). The candidates from Y. lipolytica
and Sz. pombe lack the predicted transmembrane domain and the protein from Sz. pombe
also lacks the SH3-domain.
Gic1/Gic2: These two proteins are 33% identical. Both Gic1 and Gic2 have a central PB
domain, and are probably redundantly involved in initiation of budding and cellular
polarization, interacting with Cdc42 via the Cdc42/Rac-interactive binding (CRIB)
domain. A. gossypii and K. waltii only have one such protein. Sequence conservation is
too low to allow identification of orthologues in filamentous fungi. The Gic2 orthologue
in S. kudriavzevii is significantly shorter (p=3.8*10-5), due to both annotation from a later
start, although the one from S. cerevisiae is conserved, and due to a frame shift induced
truncation, presumably resulting from a sequencing error as the downstream sequence
aligns nicely.
Rsr1: Rsr1 is a GTP binding protein of the Ras family and also part of the interaction
network of Cdc42 and Cdc24. The protein is involved in determination of cell polarity
but a role in HOG signalling has not been demonstrated. Rsr1 is well conserved (Table
5). There seem to be two Rsr1-like proteins in A. nidulans and M. grisea. The C. glabrata
and U. maydis proteins are significantly longer than their counterparts in yeasts and
filamentous ascomycetes (p=2.2*10-4 and p=3.3*10-5, respectively). Furthermore, they
are significantly larger in yeasts than in filamentous ascomycetes (26110 vs. 2174;
p=2.3*10-8, t-test).
Spa2: Spa2 is also part of the interaction network around actin and Cdc42 and it also
interacts with Ste11, but a role in HOG signalling has not been demonstrated. Spa2 is
significantly larger in yeasts (1370315 amino acids) than in filamentous fungi (94053
amino acids; P=0.00033, t-test), even excluding the gigantic A. gossypii protein (3392
residues, p=3.3*10-5). We found one candidate orthologue in each organism investigated
although identity drops below 20% in filamentous fungi. A putative Sz. pombe orthologue
has an extension that may contain transmembrane domains and it also has a number of
repeats of unknown function not present in other putative Spa2 orthologues. Together
with the fact that it is much shorter than any other Spa2 orthologue (659 residues,
p=0.009), suggests that it is a false orthologue.