The membrane-associated steps of the GET pathway − Rakyat Biologi

Yeast

Historically, the membrane requirements for TA protein targeting and insertion have been difficult to pin down. A pioneering cell-free study established that insertion of a tail-anchored SNARE protein into mammalian microsomes (ER-derived membranes) was abolished by prior protease treatment of the membrane [2]. Sec61 was excluded as a possible protease-sensitive candidate but the identities of the relevant membrane proteins were not established. Subsequently, this picture was complicated by the discovery that cytochrome b5 (Cb5) is a TA protein that inserts efficiently into liposomes (protein-free lipid bilayers) [21,22]. This could be reconciled with selective Cb5 targeting to the ER in vivo because sterols block insertion into liposomes: because the ER membrane has a relatively low sterol content compared to the other membranes in the secretory and endocytic pathways, it should be selectively permissive for Cb5 insertion. This is, however, difficult to test in vivo because the tools for manipulation of ER lipid composition are still rudimentary.

With the biochemical identification of TRC40 came the exciting possibility of using it as a molecular handle to identify the missing membrane components [23]. Indeed, Get3 had already been genetically and physically linked to two ER membrane proteins, Get1 and Get2 [24,25]. It was not long before a powerful combination of yeast genetics, cell microscopy, and cell-free insertion assays explained the strong ER selectivity of TA proteins [26]: most TA proteins interact with Get3 and are targeted to the ER through the Get3 interaction with its ER receptor, Get1/2 (Figure 1). Thus, after some historical uncertainty, the targeting of most TA proteins fell in line with the tenets of the signal hypothesis [27]: a cytosolic targeting factor recognizes a hydrophobic signal on the substrate and delivers it to the appropriate membrane by interacting with a receptor.

Three recent studies of Get1/2 interactions with Get3 have provided a clear mechanistic insight into one of the least understood steps of the signal hypothesis: how the targeting factor lets go of its substrate. Get1 and Get2 are three-pass proteins that interact via their transmembrane regions and use distinct cytosolic domain (CD) structures to bind individually to Get3 [13,28,29]. Specifically, X-ray crystallography revealed that the very N-terminal region of Get2, which is part of an otherwise intrinsically disordered cytosolic region, forms a complex with a Get3 dimer that is stabilized in the closed conformation by the presence of bound nucleotide [28,29]. This interaction is mediated by a pair of short, charged Get2 alpha helices, separated by a linker, which interact symmetrically with residues largely restricted to one or the other subunit of the Get3 dimer. Notably, Get2 binding is compatible with an assembled hydrophobic groove on Get3, which suggests that Get2 captures Get3-TA protein complexes from the cytosol with a long-reaching tether (Figure 4). This view also agrees with the conformational state of Get3 in the crystal structures of the Get3-Get1CD complex. In those studies, despite inclusion of nucleotide, the Get3 dimer is either fully or partially open and lacks nucleotide [28,29]. This is because the two Get1 coiled coils progressively shuck open the Get3 dimer and, as a result, break apart the substrate-binding groove and nucleotide-binding sites (Figure 4).

Biochemical reconstitution experiments support the substrate-release mechanism gleaned from the structures and further demonstrate that Get1 and Get2 are the minimal membrane machinery for TA protein insertion [13,28]. Namely, Get3 can still hold its substrate while interacting with Get2’s N-terminal helix-linker-helix motif, but Get1CD binding to Get3-TA protein complexes causes substrate release. Importantly, both in vivo and under physiologically relevant in vitro conditions, efficient TA protein insertion requires Get3 binding to both cytosolic domains of the Get1/2 receptor. This is because, in the absence of the Get2CD tether, the local concentration of a Get3-TA protein complex near Get1CD is not high enough to cause substrate release.

Lastly, the role of the Get3 ATP cycle in coordinating the pre-targeting and membrane-associated steps of the GET pathway is slowly coming into light. There are two crucial observations. First, a Get3-TA protein complex formed in the presence of ATP has to undergo hydrolysis before TA protein release can occur [13,28]. Second, ATP induces dissociation of Get3 from the membrane, which resets the GET pathway to its starting point [13,28] (Figure 3). Consistent with this latter finding, ATP (but not ADP or AMP) competes with Get1CD for Get3 binding. The intimate nature of this competition is illustrated by the conserved loop at the tip of Get1’s coiled coil that extends into the nucleotide-binding pocket of Get3 [28,29]. A fuller understanding of how the GET pathway presumably avoids futile cycles of ATP hydrolysis in the absence of substrate targeting (or whether it uses energy for substrate proofreading) will require a more detailed kinetic analysis. Nonetheless, we have already reached a good understanding of how an ATPase molecular switch coordinates the targeting and insertion stages of the GET pathway.

Mammalian

We know comparatively little about how TRC40 delivers TA proteins to the ER of mammalian cells, but several lines of evidence point to a conserved mechanism. First, WRB (tryptophan-rich basic protein) has weak sequence homology and a similar predicted topology to Get1 [26]. This protein localizes to the ER membrane, where it functions as the TRC40 receptor [30]. Second, the isolated WRB coiled coil forms a complex with TRC40 and inhibits TA protein insertion in vitro [30]. Third, native microsomes recruit but fail to induce substrate release from TRC40-TA protein complexes when ATP hydrolysis is blocked [4]. Last, ATP stimulates dissociation of TRC40 that co-purifies with microsomes [4].

An important piece of evidence that is still missing is whether WRB is necessary for TA protein biogenesis in vivo. Deletion of the GET1 gene in yeast cells results in cytosolic aggregation or mitochondrial mislocalization of secretory pathway TA proteins [26]. Future studies should establish if WRB RNAi knockdown leads to a similar phenotype or if increased TA protein degradation dominates under these conditions [19,31]. Furthermore, the Get2 homolog remains elusive. This is perhaps not surprising given that the majority of the Get2 cytosolic domain is intrinsically disordered and poorly conserved even among closely related yeast species. Thus, the functional requirements for the mammalian equivalent might be unimposing: a TRC40 tether (ie., a helix-linker-helix motif at the end of an intrinsically disordered cytosolic region), and transmembrane domains that interact with WRB. More sophisticated bioinformatic approaches and purification of native WRB complexes should facilitate identification of mammalian Get2, which would nail the point that the GET pathway is conserved from beginning to end.

Another issue is the recent connection between the mammalian GET pathway and ER targeting of secretory peptides. Because of their short length (<100 amino acids), these proteins are synthesized faster than the SRP pathway can target them to the ER membrane, but they still depend on Sec61 for translocation into the ER lumen [32,33]. Surprisingly, TRC40 can recognize secretory peptides in vitro and promote their ER translocation by a WRB-dependent mechanism [34]. These findings raise the intriguing possibility that the GET pathway can feed certain substrates to the Sec61 channel.