In this study, we present evidence demonstrating that Yet1p and Yet3p are required for derepression of INO1
. Cells harboring yet1
Δ and yet3
Δ mutations displayed comparable inositol-starvation growth phenotypes (Wilson and Barlowe, 2010
) that were similar in magnitude to that of the known inositol derepression mutants scs2
Δ and ire1
Δ (Cox et al.
; Kagiwada and Zen, 2003
expression during inositol starvation was decreased in a yet3
Δ mutant, as assessed by Ino1p-HA immunoblot and by an INO1-LacZ
transcriptional reporter assay. Consistent with a defect in INO1
derepression, we found that a significant amount of Opi1p remained in the nucleus during inositol starvation in a yet3
Δ mutant. Furthermore the Yet1p–Yet3p complex interacted with Scs2p and Opi1p in manner that was stimulated by inositol starvation. Disruption of the Yet1p–Yet3p complex by yet1
Δ mutation prevented Yet3p from interacting with Scs2p and Opi1p, connecting the inositol auxotrophy of a yet1
Δ mutant to the loss of Yet complex association with Scs2p–Opi1p (similar results were obtained for Yet1p in a yet3
Δ mutant, unpublished data). Together with our previous findings (Wilson and Barlowe, 2010
), these results indicate that the Yet1p–Yet3p complex directly regulates Scs2p–Opi1p-mediated INO1
We note that, whereas 2μ overexpression of SCS2
partially rescued yet3
Δ (), similar overexpression of YET1
had no detectable suppressive effect on an scs2
Δ mutant (unpublished data). These results are consistent with a model in which the Yet complex confers inositol prototrophy through Scs2p. In other words, excess Yet complex provides no benefit in the absence of SCS2
. It seems paradoxical then that an scs2
Δ mutant displays a more modest growth phenotype than a yet3
Δ mutant at 30°C and that the yet3
Δ double mutant exhibits a clear synthetic defect in the absence of inositol. However, yeast contains a homologue of SCS2
, known as SCS22
, that has been reported to play a minor role in phospholipid metabolism because an scs2
Δ double mutant showed a synthetic growth defect in the absence of inositol (Loewen and Levine, 2005
). Thus one explanation for this paradox is that although Scs22p does not notably influence the association of the Yet complex with Opi1p in the absence of Scs2p (Supplemental Figure 4), it may possess a basal level of derepression activity that requires the Yet complex but is not significantly enhanced by its overexpression.
The observation that Yet1p–Yet3p interactions with Scs2p and Opi1p were increased by inositol starvation suggests that this association facilitates Scs2p-mediated INO1
derepression. It does not seem to be simply a matter of modulating the association between Scs2p and Opi1p because the yet1
Δ mutation did not detectably affect this interaction, at least under the detergent solubilization conditions we tested. However, the observation that Opi1p was not correctly localized to the ER in a yet3
Δ mutant during inositol starvation suggests a role for the Yet1p–Yet3p complex in this process. Our favored model is that the Yet proteins bind to and sequester Scs2p–Opi1p complexes at ER membranes during inositol starvation, thus preventing diffusion and transport to the inner nuclear membrane (INM) and subsequent Opi1p-mediated repression of UASINO
genes. Previous studies have shown that, in the absence of inositol, Opi1p binds to Scs2p and an increased pool of ER-localized phosphatidic acid to remain outside the nucleus (Loewen et al.
). While this study was under review, two genome-wide screens of the yeast deletion collection identified >200 genes that influence growth rates in the absence of inositol, including yet1
Δ and yet3
Δ (Villa-Garcia et al.
; Young et al.
). Interestingly, several of these gene deletions cause reductions in intracellular pH, which decreases binding of Opi1p to phosphatidic acid and prevents efficient sequestration of Opi1p (Young et al.
). The Yet1p–Yet3p complex does not appear to influence intracellular pH but may act to further stabilize the association between Opi1p-Scs2p and the pool of ER-localized phosphatidic acid to ensure stringent derepression during inositol starvation.
Alternatively, the Yet complex could play a more active role in the movement of Scs2p–Opi1p. The mechanism by which Opi1p exits the nucleus upon inositol starvation has not been described. ER localization of Opi1p does not depend on the major nuclear export receptors (Loewen et al.
), and it has been suggested that Opi1p binds Scs2p on the INM (Brickner and Walter, 2004
). One possibility is that Opi1p exits the nucleus in complex with Scs2p. If this is the case, Yet1p–Yet3p may somehow facilitate the INM to ER relocalization of Scs2p–Opi1p during inositol starvation. These models are consistent with observations in mammalian systems showing that the Yet protein homologue BAP31 influences localization of specific substrate molecules (e.g., Annaert et al.
; Paquet et al.
; Szczesna-Skorupa and Kemper, 2006
). A final possibility is that the Yet1p–Yet3p complex facilitates Scs2p-mediated nuclear membrane recruitment of INO1
, a requisite part of its activation (Brickner and Walter, 2004
; Brickner et al.
). Remarkably, artificial recruitment of the INO1
locus to the nuclear membrane bypassed the SCS2
requirement in the inositol-starvation response (Brickner and Walter, 2004
). In contrast, this strategy was not sufficient to overcome the inositol requirement of a hac1
The Yet complex is required for Scs2p–Opi1p function although the Yet1p and Yet3p proteins appear to possess distinct functional properties. A similar relationship was reported for the mammalian homologues BAP29 and BAP31, which perform overlapping but nonidentical functions (Ladasky et al.
; Abe et al., 2009
). We observed that inositol starvation has a more significant effect on Yet1p association with Scs2p and Opi1p (clearly apparent for Opi1p-MYC coimmunoprecipitation; ) when compared with Yet3p association with Scs2p and Opi1p. Moreover, 2μ overexpression of YET1
in the presence of inositol elevated basal INO1
promoter activity, whereas 2μ overexpression of YET3
reduced this promoter activity (Supplemental Figure 1). Taken together, these results suggest that increasing the relative amount of Yet1p in association with Yet3p positively influences the ability of the Yet complex to act in INO1
We previously reported genetic and physical interactions between the Yet1p–Yet3p complex and the Sec translocation complex (Wilson and Barlowe, 2010
). More specifically, yet1
Δ and yet3
Δ mutants displayed synthetic growth defects in the absence of inositol when combined with sec63–1
, or sec71
Δ mutations. To test whether these genetic relationships are shared with other mutations in the INO1
derepression pathway, we examined the growth of scs2
Δ double mutant strains (Supplemental Figure 5). Interestingly, both scs2
Δ mutant strains showed synthetic growth defects in the absence of inositol. These findings indicate some type of a connection between Scs2p-mediated INO1
derepression and the ER translocation apparatus. We also considered the possibility that a general reduction in phosphatidyl inositol (PI) levels was not well tolerated by translocation-defective cells because PI levels are known to be diminished when cells are starved for inositol (Chang et al.
) and are presumably reduced further in yet
Δ and scs2
Δ mutants. However, this possibility seems unlikely because the ire1
Δ mutation, which also attenuates INO1
derepression (Cox et al.
), did not display synthetic growth defects in the absence of inositol when combined with sec71
Δ (Supplemental Figure 5). These results support a specific genetic connection between YET1
, and SCS2
with components of the ER translocation apparatus.
In this report, we demonstrate a direct role for the yeast BAP31 homologues in regulation of Scs2p–Opi1p-mediated derepression of INO1
. Scs2p belongs to a conserved family of ER-localized VAPs, which interact with a variety of intracellular proteins often through binding to a FFAT motif as found in Opi1p (Lev et al., 2008
). Many FFAT motif–containing proteins, including oxysterol binding proteins and ceramide transport proteins, functionally interact with VAPs to regulate lipid synthesis and transport in animal cells (Wyles et al.
; Kawano et al.
). Interestingly, a mutation in one of the human VAP genes produces late-onset familial motor neuron disease (Nishimura et al.
). On the basis of sequence conservation and preserved ER localization for the BAP31 and VAP families, we speculate that Yet1p–Yet3p regulation of Scs2p–Opi1p function in yeast represents a conserved regulatory module that controls lipid synthesis and/or transport in other eukaryotic species. Further mechanistic dissection of this pathway in yeast should contribute to our general understanding of BAP31- and VAP-controlled processes with potential connections to human disease.