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The tryptophan rich basic protein/calcium signal‐modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail‐anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous. CAML contains an N‐terminal basic, TRC40/Get3‐interacting, region, three transmembrane segments near the C‐terminus, and a poorly conserved region between these domains. Here, I searched the NCBI protein database for remote CAML homologs in all eukaryotes, using position‐specific iterated‐basic local alignment search tool, with the C‐terminal, the N‐terminal or the full‐length sequence of human CAML as query. The N‐terminal basic region and full‐length CAML retrieved homologs among metazoa, plants and fungi. In the latter group several hits were annotated as GET2. The C‐terminal query did not return entries outside of the animal kingdom, but did retrieve over one hundred invertebrate metazoan CAML‐like proteins, which all conserved the N‐terminal TRC40‐binding domain. The results indicate that CAML homologs exist throughout the eukaryotic domain of life, and suggest that metazoan CAML and yeast GET2 share a common evolutionary origin. They further reveal a tight link between the particular features of the metazoan membrane‐anchoring domain and the TRC40‐interacting region. The list of sequences presented here should provide a useful resource for future studies addressing structure‐function relationships in CAML proteins. [ABSTRACT FROM AUTHOR]

C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361–366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance. [ABSTRACT FROM AUTHOR]

Tail‐anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein‐free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5‐ER and b5‐RR, which differ only in the charge of the C‐terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell‐free and in cellula results, we analyzed targeting in digitonin‐permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C‐terminal negative charge of b5‐ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI) reduced insertion of b5‐ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C‐terminal charge, and reveal a novel, substrate‐specific ER‐targeting pathway. [ABSTRACT FROM AUTHOR]