Proteins destined for the endomembrane system of eukaryotic cells are typically translocated into or across the membrane of the endoplasmic reticulum and this process is normally closely coupled to protein synthesis. However, it is becoming increasingly apparent that a significant proportion of proteins are targeted to and inserted into the ER membrane post-translationally, that is after their synthesis is complete. These proteins must be efficiently captured and delivered to the target membrane, and indeed a failure to do so may even disrupt proteostasis resulting in cellular dysfunction and disease. In this review, we discuss the mechanisms by which various protein precursors can be targeted to the ER and either inserted into or translocated across the membrane post-translationally. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling.

The highly conserved target of rapamycin complex 1 (TORC1) is the central component of a signaling network that couples a vast range of internal and external stimuli to cell growth, proliferation and metabolism. TORC1 deregulation is associated with a number of human pathologies, including many cancers and metabolic disorders, underscoring its importance in cellular and organismal growth control. The activity of TORC1 is modulated by multiple inputs; however, the presence of amino acids is a stimulus that is essential for its activation. Amino acid sufficiency is communicated to TORC1 via the highly conserved family of Rag GTPases, which assemble as heterodimeric complexes on lysosomal/vacuolar membranes and are regulated by their guanine nucleotide loading status. Studies in yeast, fly and mammalian model systems have revealed a multitude of conserved Rag GTPase modulators, which have greatly expanded our understanding of amino acid sensing by TORC1. Here we review the major known modulators of the Rag GTPases, focusing on recent mechanistic insights that highlight the evolutionary conservation and divergence of amino acid signaling to TORC1.

SUMMARY The endomembrane system of yeast contains different tail-anchored proteins that are posttranslationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment may be hazardous in the cytosol if membrane insertion fails resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function; promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.

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