Some outstanding questions about the GET pathway

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Is the ribosome a conserved component of the GET pathway?  Proteomic analysis identified Get5 as a ribosome-associated protein [35] but more directed biochemical experiments should establish if the yeast GET pathway, like the mammalian one, also begins on the ribosome. Evidence for a conserved ribosome surveillance mechanism by the GET pathway would be exciting because yeast genetics could then be used to hunt for ribosomal get mutants. More concretely, a mammalian in vitro system should be used to establish whether TA proteins are mistargeted when the connection between the ribosome and the Bag6 complex is severed. Lastly, cryoelectron microscopy studies should look for meaningful structural changes in the ribosome that track with the ability of nascent chain sequences in the exit tunnel to generate a recruitment signal for the Bag6 complex.

What is the role of Sgt2/SGTA-tethered heat shock proteins (Hsps)? Prior to the discovery that SGTA directly recognizes tail anchors, many studies established that it also co-chaperones Hsp-mediated protein folding of non-TA protein substrate [36,37,38]. The interaction between Sgt2 and Hsps, however, is not apparently crucial for efficient TA protein capture and handoff [8,11]. Nonetheless, a possible connection between Hsps and the GET pathway might still exist. Specifically, protein localization by fluorescence cell microscopy of get1/2 mutants revealed that Get3 is recruited to TA protein aggregates in a Get4/5-dependent manner [39]. Thus, establishing if Sgt2/SGTA and/or the associated Hsps go after TA protein aggregates to maintain protein homeostasis is an important future goal.

What is the mechanism of the insertion step? It is not known how tail anchors get inserted into the membrane after they are released from Get3. One proposal is that this occurs spontaneously [21] (Figure 5). In this view, the GET pathway is done with the tail anchor once it deposits it on the cytosolic surface of the ER membrane. Indeed, moderately hydrophobic peptides spontaneously insert into liposomes all on their own [40]. Most tail anchors are significantly more hydrophobic, however, which is why the GET pathway is necessary to maintain them in a soluble state and, at the same time, confer selectivity for the ER membrane. Thus, an alternative hypothesis is that the GET pathway assists the insertion step [4] (Figure 5) presumably to reduce a kinetic barrier to spontaneous insertion. Spontaneous insertion models for protein targeting pathways are intrinsically difficult to prove and have historically been in decline [41]. Future studies of the structure and function of Get1/2 transmembrane domains should reveal whether another spontaneous insertion model bites the dust.