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Abstract: A large group of diverse, functionally important, and differently localized transmembrane proteins, share a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. The C-terminal membrane anchor of these tail-anchored (TA) proteins generally represents the sole targeting determinant, and becomes available to targeting factors only after release of the finished polypeptide from the ribosome. Hence, TA proteins do not have a chance to interact co-translationally with Signal Recognition Particle and are delivered post-translationally to all target membranes, including the ER. Recent work has demonstrated the existence of different biogenetic pathways for TA proteins. Notably, some are able to efficiently translocate their C-terminus across protein-free bilayers without the participation of any membrane or cytosolic protein, while others require assistance from cytosolic chaperones and membrane receptors. In this review, we summarize current knowledge on the different insertion pathways, with emphasis on a recently discovered chaperone system that operates in fungi as well as in higher eukaryotes to deliver TA proteins to the ER (called Guided Entry of Tail-anchored Proteins (Get) system and Transmembrane Recognition Complex (TRC), in yeast and mammals, respectively). We suggest that the final insertion step of TA proteins does not require membrane proteins, but that different competing chaperone systems ensure precise delivery to defined targets while preventing inappropriate insertion into otherwise permissive bilayers. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes. [Copyright &y& Elsevier]

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/ Get2 heterotetramer and in mammals of theWRBprotein (tryptophan- rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenousWRBand CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40- associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ~5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRBprotein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excessCAMLin the mammalian ER. [ABSTRACT FROM AUTHOR]