The present study defines a pathway by which cells ensure the efficient and accurate biogenesis of TA proteins destined for the secretory pathway. The soluble cytosolic ATPase, Get3, specifically queries newly synthesized proteins for the presence of C-terminally localized hydrophobic domains. Get1 and Get2 then serve as an ER membrane receptor, which recruits the Get3-TA complex, thereby promoting the proper insertion of TA proteins into the ER (A). Once inserted, TA proteins can then be routed to their ultimate destination within the secretory pathway. In the absence of the heteromeric Get1/Get2 receptor, TA proteins bound to Get3 fail to reach ER membranes, and are instead trapped in large cytosolic aggregates (B). This leads to a broad depletion of TA proteins, which in turn can account for the otherwise confusing array of phenotypes associated with loss of Get proteins. Binding to Get3 is also a decisive step in the insertion pathway, as in its absence, secretory pathway TA proteins may insert into mitochondrial membranes (C).
Schematic Model for GET Complex Function
The finding that the GET pathway is not essential for yeast viability provides in vivo support to in vitro studies that had suggested additional mechanisms by which TA proteins can find their destination membranes (Rabu and High, 2007
). Nonetheless, several considerations suggest that the GET pathway is the major route used to target a broad range of TA proteins to the secretory pathway. First, our Y2H analysis indicates that Get3 can bind multiple secretory pathway TA proteins in a TMD-dependent manner. Second, for all secretory pathway TA proteins examined, the interaction with Get3 caused sequestration of the TA proteins into cellular aggregates in the absence of Get1 and Get2. This suggests that, when Get3 is present, most of the natural flux of TA proteins flows through the GET pathway. Indeed, yeast fail to grow when Get3 is overexpressed in the absence of Get1 and Get2 (Figure S4
). Third, deletion of Get3, which would eliminate the GET pathway without actively preventing TA proteins from utilizing alternate pathways by trapping them in nonproductive Get3 complexes, still leads to diverse cellular defects. Finally, in vitro reconstitution experiments directly establish that Get3 cooperates with the Get1/2 complex in mediating the insertion of newly synthesized TA proteins. Thus the ability of cells to survive in the complete absence of the Get proteins may be analogous to the viability of yeast missing the SRP, which is made possible by the existence of alternate pathways for insertion of the numerous secreted and membrane-bound proteins that normally utilize this machinery (Ogg et al., 1992
Possible alternate routes for TA protein biogenesis that have been suggested by in vitro studies include spontaneous insertion, which occurs efficiently for some TA proteins, such as CytB5 (Brambillasca et al., 2006
). In addition, purified Hsc70/Hsp40 can promote the ATP-dependent (Abell et al., 2007
) and SRP the GTP-dependent insertion of other TA proteins, such as Sec61β, (Abell et al., 2004
). Such back-up systems, however, would lack the strong membrane specificity conferred by the ER localization of the Get1/2 complex, as well as the preferential binding of Get3 to TA proteins destined to the secretory pathway. The potential importance of such specificity is illustrated by the observation that some TA proteins, including Pex15 and Ubc6, mislocalize to the mitochondria when the GET system is impaired. This argues that, shortly after synthesis, Get3 competes with other factors (possibly Hsc70 and/or components that play an analogous role to Get3 in the targeting of mitochondrial TA proteins) for TMD binding, and that Get3 recognition commits the TA proteins to their subsequent insertion into ER membrane. It remains to be determined whether a dedicated protein machinery exists that ensures the accurate targeting of mitochondrial TA proteins, or whether the shorter, more hydrophilic nature of their TMDs prevents Get3 binding, thereby allowing for efficient, spontaneous insertion into the mitochondria.
The interaction between Get3 and a TA protein substrate may thus represent a critical and potentially regulated decision step for establishing the destination target of TA proteins. Regulation could globally alter Get3 function or specifically affect the interaction between Get3 and target TA proteins. Along these lines, we have recently found that the function of Get3 is modulated by its redox state (our unpublished data and Metz et al. 
). In addition, Get3 is transcriptionally upregulated under both cytosolic (Auld et al., 2006
) and ER (Travers et al., 2000
) stress conditions. It has also been found that many TA proteins are palmitoylated (Roth et al., 2006
) or phosphorylated (such as for Sed5 [Weinberger et al., 2005
]) on residues that are immediately adjacent to the TMD. Such modifications could modulate Get3 recognition by creating negatively charged flanking regions or by altering the hydrophobicity of the TMD, thereby enabling the coordinated regulation of subclasses of TA proteins and altering the physiology of the cell.
While the present studies focused on TA biogenesis in yeast, recent observations suggest that the GET pathway plays an essential role in TA biogenesis in higher eukaryotes. Biochemical studies revealed that the mammalian Get3 homolog, Asna1/TRC40, binds the TA protein, Sec61β, and facilitates its posttranslational insertion into ER membranes (Stefanovic and Hegde, 2007; Favaloro et al., 2008
). An in vivo role of Asna1 in TA biosynthesis in metazoans is suggested by the impaired capacity for insulin secretion in Caenorhabditis elegans
mutants of asna1
(Kao et al., 2007
). In light of our findings, an attractive hypothesis is that impaired insulin secretion results from compromised biogenesis of one or more of the SNARE TA proteins. The broader importance of the GET pathway is underscored by the finding that complete loss of ASNA1
causes early embryonic lethality in mice (Mukhopadhyay et al., 2006
) and arrested growth at the L1 stage in C. elegans
(Kao et al., 2007
). The molecular identity of the Get3 ER receptor in metazoans remains to be established. However, we find that Ysy6 translated in rabbit reticulocyte extracts inserts into yeast microsomes in a Get1/2-dependent manner, suggesting that the GET pathway is highly conserved (data not shown). Consistent with this, PSI-BLAST analysis identifies the WRB protein as an excellent and ubiquitously expressed candidate for a Get1 ortholog.
In summary, the GET complex in yeast and likely metazoans constitutes the major machinery necessary for membrane selective, and ATP-dependent insertion of TA proteins. This finding should now enable mechanistic studies to explore central questions, including how the GET system selects substrate and exploits ATP hydrolysis to overcome the energetic barriers to insertion of transmembrane proteins into lipid bilayers.