Gcn1 and Actin Binding to Yih1

Abstract

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68 -258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1-and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68 -258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.In all eukaryotes, phosphorylation of the ␣-subunit of translation initiation factor 2 (eIF2␣) 5 is a major mechanism for the regulation of protein synthesis in response to a variety of environmental or intracellular stresses. This event leads to general translation inhibition at the same time that it allows for increased translation of specific messages, such as GCN4 in yeast and ATF4 in mammals. These code for transcription factors that activate a network of genes aimed at cell recovery from the initial stress (1).Gcn2, the sole eIF2␣ kinase in the yeast Saccharomyces cerevisiae, is activated by amino acid starvation and imbalance and other stresses (2). Gcn2 is a highly conserved protein found in all eukaryotic organisms, and in mammals it has been implicated in additional functions such as modulating the immune system, feeding behavior, and memory formation (3-6). Gcn2 is composed of five distinct functional domains. At its N terminus, a region called the RWD domain (from its presence in RING finger proteins, WD-repeat-containing proteins, and yeast DEAD-like helicases) (7) binds directly to the activator protein Gcn1 (8, 9). Its kinase catalytic domain is found in the center of the protein. Between the RWD domain and the kinase domain resides a pseudo-kinase domain, identifiable by characteristic but incomplete protein kinase sequences, with no well defined function. A d...

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