Mechanism of precise intracellular targeting of spontaneously inserting tail-anchored proteins

Background: Tail-anchored (TA) proteins are a subclass of membrane proteins that carry out several functions and that are targeted to their destination by unique post-translational pathways. Some TA proteins can insert in vitro into pure phospholipid bilayers spontaneously, i.e, without assistance from any chaperone, while others depend on the Get3/TRC40 pathway. One example of a spontaneously inserting TA protein is cytochrome b5, of which two forms are known: one targeting the Endoplasmic Reticulum (b5-ER) and the other one targeting the Mitochondria Outer Membrane (b5-RR). Observations: Microinjection of the recombinant b5 proteins into cells results in precise targeting of each of the two proteins, indicating that the targeting information is present in the protein. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate (RRL), we obtain faithful targeting for both forms of cyt b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. Attempting to find the energy-dependent chaperone responsible for the ER targeting, we observed that TCR40, Hsc-70 and Snd2 pathways play only a modest role. Recently, we found that Eyearestatin I, a p97 inhibitor, strongly inhibits the delivery of b5-ER to the ER, as monitored by glycosylation of an engineered N-glycosylation site near the C-terminus, while having no effect on a TRC40-dependent substrate. Hence, the effect seems to be specific for spontaneously inserted tail-anchored proteins, but the exact mechanism is still under investigation. Conclusions: Our results demonstrate that cyt b5 is precisely targeted to the ER by pathways distinct from the one mediated by Get3/TRC40. Since interference with TRC40 and Snd2 only minimally affect b5 targeting, it appears that multiple redundant mechanisms underlie the final specific targeting of this spontaneously inserting TA protein.