Discussion

The study done by Krogan et al. (2006) identified the majority of the potential YBP of all published large-scale interactome studies (cf. Table C2). This is partly because they provided low scoring interactions as supplementary data.

Chapter C. Identification of potential Yih1-binding proteins (YBP)

64 Despite this, they did not identify the known Yih1-binding protein actin while Gcn1 was only found indirectly in a complex with other YBP. One explanation might that the TAP tagging method that they used is more likely to miss weak or transient interactions because of the two-step procedure which leads to a high dilution of the protein solutions in the later steps (Gavin et al., 2002; Tagwerker et al., 2006; Xu et al., 2012).

Some proteins co-precipitated more than once with Yih1, either directly with TAP-Yih1 or indirectly with one of the tagged proteins that co-precipitated Yih1 (cf. Figure C2). These are strong candidates for novel YBP. As they are found relatively frequently in a complex with Yih1 compared to other proteins they are more likely be YBP or proteins that are involved in Gcn2 regulation.

Both Krogan et al. (2006) and Tarassov et al. (2008) found the known Yih1-binding protein Gcn1 and, of all proteins that were found in a complex with Yih1, this is the only protein that was identified in more than one study. Curiously, the proteins Jsn1, Pir5 and Urh1 were only found as Yih1 interactors by the two yeast two-hybrid studies (Uetz 2000, Ito 2001). They were not found in the affinity purification studies, not even as low scoring proteins. These may suggest that both yeast two-hybrid approaches identified different subsets of protein-protein interactions or a high number of false positives. On the other hand, the affinity studies may show a large coverage but at the same time a low sensitivity and thus might have missed these interactors. These findings are an indication that not all Yih1-binding proteins have been found so far.

One known caveat with affinity assays is that they cannot be used to determine if there is a direct physical interaction between proteins (Mackay et al., 2007, 2008). A co-precipitating protein may not directly interact with Yih1 but instead it may be connected via one or more bridge proteins. In addition, there is the possibility of false positives due to the formation of protein complexes that may not exist in a native environment or due the inclusion of promiscuous proteins. Other methods such as the yeast two-hybrid system may be more informative in regards to determining direct interactions (Yu et al., 2008). However, a YBP that does not directly interact with Yih1 may still be involved in promoting Gcn2 function, for example via a mediating protein (Figure A8).

Chapter C. Identification of potential Yih1-binding proteins (YBP)

65 Proteins that are not known to bind Yih1 but are involved in the GAAC were identified. Only Gcn3 (eIF2Bα) (Hannig and Hinnebusch, 1988) and eEF1A (Visweswaraiah et al., 2011b) were found in a complex with a tagged Yih1 while Gcn1, Gcn2, Gcn20, eEF3 and eIF2α were found only indirectly with Yih1 by co-precipitating with other tagged YBP. Gcn2 and Gcn20 might be expected to co-precipitate because they are binding Gcn1 and indeed, they were only found in a complex that also contained Gcn1. Similarly, eIF2α is binding Gcn2 and it was co-precipitating Gcn2 and Gcn1. Gcn20 and eIF2α were only found once in a complex with Yih1, suggesting a weak interaction with its respective binding partners. Other proteins that are involved in the GAAC were identified more than once with Yih1, such as eEF1A and Gcn3 (eIF2Bα). eEF1A is highly abundant and is often considered a contaminant in affinity purification studies. However, there is already evidence that it may be in a complex with Yih1 (E. Sattlegger and B. Castilho, unpublished data). In addition, as it is binding Gcn2 and is involved in the GAAC it was considered a putative YBP (Visweswaraiah et al., 2011b). Additionally, eEF3 was found in a complex with tagged Ilv1 and Yih1. This suggested that Yih1 could perform its Gcn2-inhibitory function not only by binding Gcn1 but also by binding other proteins involved in protein translation. For example, it was proposed that eEF1A may deliver uncharged tRNA to Gcn2 or that this is mediated by Gcn1 (Visweswaraiah et al., 2011b). Thus, Yih1 may inhibit this process by binding to eEF1A, Yih1 may recruit another factor, or if Gcn1 is involved then Yih1 could inhibit the ability of Gcn1 to transfer tRNA to Gcn2 (Sattlegger et al., 2004).

Three proteins were most frequently found in different complexes that contained Yih1 (Figure C2). Aap1 acts as a positive regulator of glycogen accumulation (Caprioglio et al., 1993). Under histidine starvation conditions its expression is induced by Gcn4 (Natarajan et al., 2001), indicating that it has a role in the GAAC. However, as this role is downstream of Gcn4 it is likely not important for Gcn2 function, although the possibility that it may be a YBP but without regulating Gcn2 cannot be excluded. Pop2 is involved in mRNA degradation (Daugeron et al., 2001). Degradation of mRNA and translation are in a balance under replete conditions. In the case of amino acid starvation the level of mRNA for proteins involved in the starvation response is increased (Gasch et al., 2000). It

Chapter C. Identification of potential Yih1-binding proteins (YBP)

66 is conceivable that when a cell senses the starvation the activity of Pop2 (and other factors) is increased in order to recycle mRNA for the synthesis of mRNA for starvation response proteins. As Yih1 is repressing the starvation response it may act as a Pop2 inhibitor under replete conditions by binding to it and is released under starvation conditions. As strains without Yih1 do not show a growth defect this may involve other factors or Yih1 is only involved under certain conditions or to a lower extent in this pathway. Shp1 is involved in mitosis and is associated with the proteasome due to its ability to bind ubiquitylated proteins. Interestingly, Shp1 positively regulates Glc7, a protein phosphatase that acts in opposition to Gcn2 in that it reduces eIF2α phosphorylation (Wek et al., 1992). Thus, a Yih1-mediated activation of Shp1 may allow a reduction of the GAAC by reducing the level of eIF2α-P.

In summary, half of the large-scale protein-protein interactions studies have not identified Yih1 either as a co-precipitating protein in affinity studies or as an interactor in yeast two-hybrid and protein complementation assays. Between all five studies that identified YBP there was no overlap in identified putative Yih1-binding proteins. In addition, the known YBP actin and Gcn1 were not found or were in a complex with Yih1 only indirectly, respectively. This underscored the incompleteness of the Yih1 interactome.

Chapter C. Identification of potential Yih1-binding proteins (YBP)

67