The pre-targeting steps of the GET pathway

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Yeast

Entry of newly synthesized TA proteins into the GET (guided entry of TA proteins) pathway in Saccharomyces cerevisiae begins with efficient TMD capture by Sgt2 (a small glutamine-rich tetratricopeptide repeat-containing protein) [8] (Figure 2). This chaperone shields TMDs after they are released from the ribosome to prevent TA protein aggregation in the cytosol or mistargeting to mitochondria [9,10,11]. Sgt2 is in a complex with Get4 and Get5, two pathway components that facilitate TA protein transfer from Sgt2 to Get3, a homodimeric ATPase that is the ER membrane targeting factor of the GET pathway [8,11].  This is achieved by a dual mechanism (Figure 2). First, ATP stimulates binding of Get3 to Get4 [12,13,14], and this increases the local concentration of Get3 near TA proteins because of the Get4-Get5-Sgt2 bridge [8]. Second, Get4 increases the intrinsic rate of Get3-TA protein complex formation, most likely by making the Get3 dimer conformation receptive for TMD binding [8] (Figure 2). These biochemical insights agree with the dominant structural view of TMD recognition by Get3 [15]: ATP binding converts the Get3 dimer from an open to a semi-closed state; ATP hydrolysis fully closes the Get3 conformation, creating a composite, hydrophobic groove that most likely cradles the TMD. In addition, there is some evidence for a competing view that tail anchors are sandwiched inside a tetrameric Get3, which has a head-to-head arrangement of hydrophobic grooves [16].  Regardless of the precise structural details, the sum of biochemical and structural evidence has revealed a surprisingly elaborate pre-targeting mechanism that facilitates TMD recognition by Get3.

Mammalian

A similar pre-targeting mechanism is also required for delivering mammalian TA proteins to TRC40 (the Get3 homolog; originally named the 40kDa component of the TMD recognition complex). In this case, Bag6 (a protein encoded by BCL2-associated athanogene 6, which has no apparent yeast homolog) captures newly synthesized TA proteins [17,18] as part of a stable protein complex with the mammalian Get4 and Get5 homologs, TRC35 (C7orf20) and Ubl4A, respectively [17,19] (Figure 3). The mammalian homolog of Sgt2 (SGTA) also recognizes TMDs, but it appears to associate weakly with Bag6 [18,20]. Remarkably, even though TMD recognition by the Bag6 complex is post-translational, it can still occur on the ribosome [17]. The precise molecular details remain to be worked out, but in vitro nascent TMD sequences inside the exit tunnel enhance recruitment of the Bag6 complex to the ribosome (Figure 3). Moreover, TMD synthesis slows down mRNA translation, thus presumably allowing enough time for the Bag6 complex to be recruited before translation terminates. More concretely, following completion of proteins synthesis, TA proteins can remain ribosome-associated via the Bag6 complex and then get transferred to TRC40 (Figure 3). It would be satisfying to find that a conserved mechanism in yeast selectively recruits Sgt2 to ribosomes with nearly completed TA protein nascent chains. Certainly, this would strengthen the view that the very hydrophobic nature of most tail anchors necessitated a mechanistic convergence between the GET and SRP/Sec61 pathways, to create distinct substrate channeling mechanisms that minimize TMD exposure to the cytosol during transit from the ribosome to the ER membrane.