The natural history of Get3-like chaperones

Abstract

Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail-anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40-insert and the hydrophobic groove essential for tail-anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40-insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40-insert is present in all domains of life, we suggest that its presence does not automatically predict a tail-anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40-insert but have not been demonstrated to function in tail-anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.

Keywords: Chlorophyta; Embryophyta; Get3p; Rhodophyta; bacteria; endoplasmic reticulum; molecular chaperone; tail-anchored protein.

Figure 1

Maximum likelihood rooted phylogenetic tree of three representative sequences of each group of Get3 homologs as defined in Table 1. Percentage of trees in which the sequences clustered together after applying 1000 bootstraps are indicated at nodes if the value is higher than 70%. Scale bar indicates number of substitutions per site

Figure 2

Structure of selected Get3 homologs. Top: individual domains (A) or subunits (B and C) are marked in cyan and magenta. Bottom: hydrophobic groove or homologous region shown in surface view. Hydrophobic and nonpolar residues are shown in white, polar residues shown in green, acidic residues shown in red and basic residues are shown in blue. To allow a better view of the interior of the groove, only half of the groove is shown in surface view (B and C). A, Structure of E. coli ArsA (PDB ID: 1F48). The region unique to ArsA is highlighted in orange. Heavy metal ion coordinating cysteines are shown as ball‐and‐stick models. B, Structure of S. cerevisiae Get3 (PDB ID: 4XTR). The region homologous to the one marked in orange in A is also marked in orange here. C, Structure of Get3 from a Nostoc species (PDB ID: 3IGF). The α‐crystallin domain is depicted in red

Figure 3

Comparison of the TRC40‐insert between species. A, Known secondary structure of ScGet3 (top) compared with the predicted structure of the same region in different species (bottom, predicted helices marked with black frame). Hydrophobic residues shown in peach, aromatic residues in ochre, basic residues in blue, acidic residues in red, hydrophilic residues in green, proline and glycine in mauve, cysteine in yellow. B, Structure of M. jannaschii Get3 (PDB ID: 3UG6). Subunits are marked with cyan, magenta, orange and blue. The region homologous to the region between helix 7 and 9 in ScGet3 is shown in red. C, Distribution of the length of the region homologous to the sequence between helix 7 and 9 in ScGet3 among the sequences used for the current analysis. All bins containing at least 1% of the sequences are shown in the chart. Number of analyzed sequences: Bacteria—299; Archaea—376; Fungi—489; Animals—140; Land plants (cytoplasmic) —78; Land plants (organellar, excluding α‐crystallin domain Get3 homologs)—87. D, Distribution of the number of methionine residues in the region homologous to the sequence between helix 7 and 9 in ScGet3 among the sequences used for the current analysis. All bins containing at least 1% of the sequences are shown in the chart. The number of sequences analyzed are as in C

Figure 4

Consensus sequence and important features of the helices flanking the hydrophobic groove and of the region C‐terminally adjacent to it. A, Consensus sequence of the region homologous to ScGet3 helix 7 and 9 in different groups of Get3 homologs. Residues flanking the hydrophobic groove in ScGet3 are marked with an arrow. Residues shown to be important for TA protein binding are marked with a red arrow. Corresponding residues are highlighted with a red rectangle in the ArsA consensus sequence. B, Consensus sequence of the bottom of the hydrophobic groove (ScGet3 helix 6) in different groups of Get3 homologs. Residues facing the hydrophobic groove in ScGet3 are marked with an arrow. Heavy metal ion coordinating cysteine in ArsA and additional proline residues in organellar Get3 homologs highlighted by red boxes. C, Comparison of the CxC motif and key Get1/Get2/Get4 residues between ScGet3 and organellar homologs of Get3 in land plants. D, Distribution of the number of methionine residues in the region homologous to the sequence from helix 4 to 9 in ScGet3 among the sequences used for the current analysis. All bins containing at least 1% of the sequences are shown in the chart. Number of analyzed sequences: Actinobacteria—62; Firmicutes—47; Fungi—489; Vertebrates—70

Figure 5

Get3 homologs use various strategies to form dimers. A, Comparison of the sequence adjacent to the CxxC motif in ScGet3 and homologs from other organisms. B, Consensus sequence and secondary structure prediction of the charged C‐terminal helix found in cytoplasmic Get3 homologs in land plants. C, Graphical representation of main structural features of land plant (LP), chlorophyte (C) and red algal (R) Get3 homologs. D, Graphical representation of main structural features of bacterial Get3 homologs. E, Comparison of the presence or absence of a TRC40‐insert containing Get3 homolog in bacterial species with the number of predicted TA proteins in the given species. Empty circles represent Proteobacteria and Cyanobacteria with TRC40‐insert containing Get3 homologs arranged as two domains in a single polypeptide