The FATC domain is essential for the function of
Tra1 and other PIKK family members (Priestley
et al. 1998;
Beamish et al. 2000;
Takahashi et al. 2000;
Sun et al. 2005;
Hoke et al. 2010). For
Tra1, this is apparent from the fact that a protein containing an additional C-terminal glycine residue will not support viability (
Hoke et al. 2010). Altering the terminal phenylalanine of
Tra1 to alanine is less severe, but results in slow growth in rich media at 30° and under conditions of stress. We have shown that alleles of
TTI2 suppress
tra1-F3744A.
tti2-F328S restored all of the measured properties of the strains to ~80% of the wild-type level. Suppression by
tti2-F328S was specific for mutations in the FATC domain of
Tra1;
tra1-L3733A was suppressed, whereas alleles altering the PI3K domain, or other SAGA or NuA4 components were not. The suppression by alleles of
TTI2, whose product with
Tel2 and
Tti1 is proposed to act as a chaperone (
Horejsí et al. 2010;
Hurov et al. 2010;
Kaizuka et al. 2010;
Takai et al. 2010), the reduced levels of
Tra1 in the
tel2-15 strain, and the increased number of proteolytic products seen after Western blotting
Tra1-F3744A lead us to suggest that the FATC domain is important for
Tra1 to acquire or stabilize a fully functional conformation. The finding that the F3744A mutation increased levels of cytoplasmic
Tra1 is consistent with this model, or alternatively for roles of the FATC domain and
Tti2 in protein trafficking. Loss of any of these possible roles would deplete functional
Tra1 and would be expected to act broadly, given the importance of independent
Tra1 (Helmlinger
et al. 2011), as well as the SAGA and NuA4 complexes; indeed we find numerous phenotypic consequences of
tra1-F3744A.
The eGFP-
Tra1-F3744A present in the cytoplasm was not uniformly distributed, but appeared in foci. Though not specific for any one membrane type, we propose that these foci represent
Tra1-F3744A associated with membranes and that the altered FATC domain potentially traps these molecules on the membranes. In turn, this finding predicts that the folding of
Tra1 and perhaps the formation of some of its multisubunit complexes may occur on membranes. This is appealing because the membrane would provide a platform for the process to occur, and perhaps protect the C-terminal domains from proteolysis. A requirement for membrane interactions provides a rationale for the large number of synthetic interactions observed between membrane trafficking components and either
tra1-SRR3413 or deletions of NuA4 component genes (
Hoke et al. 2008a;
Mitchell et al. 2008). Membrane interactions are also consistent with the lipid binding properties of some of the SAGA components (
Hoke et al. 2008b). In the event of reduced complex formation the molecules could be targeted to the vacuole, perhaps providing an explanation for the
Pep4-dependent cleavage of
Spt7 (
Spedale et al. 2010). Interestingly,
Han and Emr (2011) have recently shown that
Cti6 and
Tup1 assemble with
Cyc8 on late endosomal membranes, mediated through their binding of phosphatidylinositol-3,5-diphosphate. Lipid binding is required for nuclear import of
Cti6-
Tup1-
Cyc8, interaction with SAGA, and activation of galactose-regulated genes. Membranes are inherently sensitive to many environmental cues. As
Han and Emr (2011) suggest, the membrane assembly of transcriptional complexes provides a tight link with the environmental state.
Our results in combination with the association of
Tra1 and
Tti2 determined by
Shevchenko et al. (2008) clearly indicate a functional relationship between these molecules. The connection between
Tel2,
Tti2, and
Tti1 demonstrated in mammalian cells suggests that the TTT complex is also functionally associated with
Tra1 (
Hayashi et al. 2007;
Takai et al. 2007,
2010;
Hurov et al. 2010;
Kaizuka et al. 2010). How this relates to other components of the ASTRA complex is less clear. In that it contains
Rvb1 and
Rvb2, ASTRA resembles an assembly of the R2TP (Huen
et al. 2009 and TTT complexes with
Tra1, similar to that seen for mTOR (
Horejsí et al. 2010). The potential transient nature of ASTRA and its possible role in the folding/stability of
Tra1 agrees with it not yet being isolated as an intact biochemical entity. Alternatively the suppression by
Tti2 may take place in the context of an independent TTT complex, with ASTRA required for additional functions.
tti2-F328S acts in a partially dominant fashion to suppress
tra1-F3744A. The suppression was most notable with
tti2-F328S as the sole copy of the gene, but was still apparent in the context of the wild-type allele. With the specific mechanism of the TTT complex unknown, we can only speculate on how
Tti2-F328S and
Tti2-I336S act. A strict gain of function is possible, but perhaps less so given the two alleles and the partial dominance. Alternatively, the two mutations may disrupt an interaction or property of
Tti2 that otherwise results in its inhibition. The dominant nature of the allele also suggested that the FATC domain of
Tra1 might interact closely with the region of
Tti2 surrounding F328.
Tra1-F3744A may be unable to interact, and suppression result from the restored interaction with
Tti2-F328S. The possibility of a direct interaction was attractive, given that a hydrophobic contact for the wild-type proteins could be replaced by a hydrogen bond between the serine of
Tti2-F328S and the C terminus of
Tra1. However, additional experiments were inconsistent with a direct contact. First this model would not easily explain suppression of
tra1-L3733A or the ability of
tti2-I336F to suppress. Second, if the domain of
Tti2 were to directly contact the FATC domain, one might expect negative effects on the other FATC domain containing proteins. The only discernible phenotype of
tti2-F328S in isolation was a slight slow growth in media depleted of phosphate.
Tti2-F328S did not lead to sensitivity to the DNA damaging agents MMS or phleomycin, suggesting a minimal effect on
Mec1 and
Tel1. Finally, we were unable to detect an interaction between a fragment of
Tra1 containing the PI3K and FATC domains with the C-terminal half of
Tti2 using recombinant proteins. We conclude that
Tti2-F328S enhances the activity of
Tra1-F3744A, likely by affecting folding, through a mechanism that does not restore interaction between the molecules or involve increased levels of
Tti2. We note also that
tti2-F328S does not suppress a mutation converting the terminal tryptophan of
Mec1 to alanine (
Figure S4). This suggests either that folding of the FATC domain of
Mec1 does not require
Tti2 function or that the change in function of
Tti2-F328S is specific for
Tra1.
Expression of the NuA4 (
Nourani et al. 2004) and SAGA-regulated (
Gregory et al. 1998)
PHO5 promoter was reduced approximately fivefold by
tra1-F3744A. Consistent with an effect of this mutation on NuA4 function,
Tra1-F3744A reduced histone H4 acetylation of the
PHO5 promoter. In contrast, and despite
Gcn5 being required for activated expression, the F3744A mutation had little effect on histone H3 acetylation at the
PHO5 promoter. Since the breadth of the phenotypes attributable to
tra1-F3744A suggests that SAGA function is altered, we propose that the lack of change in histone H3 acetylation is due to the ability of the Ada complex (
Eberharter et al. 1999), including
Gcn5,
Ada2, and
Ngg1, to act independently of SAGA. These results with
Tra1-F3744A are similar to what we observe at
PHO5 upon deletion of
Spt7: partially reduced acetylation, significantly decreased expression (D. Dobransky and C. J. Brandl, unpublished results). The lack of correlation between the importance of these molecules to
PHO5 expression and their effect on acetylation suggests that specific targeting of
PHO5 acetylation by SAGA is required for expression.
