Endoplasmic reticulum membrane receptors of the GET pathway are conserved throughout eukaryotes

Abstract

Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plantArabidopsis thaliana, most components of the pathway were identified throughin silicosequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors ofAtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses withAtGET1, and binds toArabidopsisGET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of otherAtgetlines, its heterologous expression together with the coreceptorAtGET1 rescues growth defects ofΔget1get2yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 inArabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

A conserved Guided Entry of Tail-anchored pathway is involved in the trafficking of tail-anchored membrane proteins inPlasmodium falciparum

“…We were unable to identify any prospective plasmodial homolog Get2/CAML in our BioID fraction based on the amino acid sequence similarity/identity. However, cross-kingdom members of Get2/CAML have recently been shown to display extreme variations in sequences; rather the members exhibited conservation in their apparent structural properties, namely: a positively charged N-terminus stretch (with at least four arginine or lysine residues in a stretch), the putative numbers of TMDs (three) and the topology of the protein (N-terminus Cytosol -TMDs-C-terminus Luminal ) [145]. We therefore mined the P. falciparum 3D7 proteome in PlasmoDB to shortlist proteins fulfilling both the criteria, i.e., (i) the presence of the RR8422 (Arg-Arg-Polar-Basic-Aliphatic-Aliphatic) motif (as a representation of the conserved RRRK motif commonly seen in CAMLs; [146]) within the first 50 amino acids from the N-terminus, and (ii) the presence of at least 3 TMDs ( Figure 6A ).…”

A conserved guided entry of tail-anchored pathway is involved in the trafficking of a subset of membrane proteins in Plasmodium falciparum

“…We were unable to identify any prospective plasmodial homolog Get2/CAML in our BioID fraction based on the amino acid sequence similarity/identity. However, cross-kingdom members of Get2/CAML have recently been shown to display extreme variations in sequences; rather the members exhibited conservation in their apparent structural properties, namely: a positively charged N-terminus stretch (with at least four arginine or lysine residues in a stretch), the putative numbers of TMDs (three) and the topology of the protein (N-terminus Cytosol -TMDs-C-terminus Luminal ) [ 126 ]. We therefore mined the P .…”

“…We were unable to identify any prospective plasmodial homolog Get2/CAML in our BioID fraction based on the amino acid sequence similarity/ identity. However, cross-kingdom members of Get2/CAML have recently been shown to display extreme variations in sequences; rather the members exhibited conservation in their apparent structural properties, namely: a positively charged N-terminus stretch (with at least four arginine or lysine residues in a stretch), the putative numbers of TMDs (three) and the topology of the protein (N-terminus Cytosol -TMDs-C-terminus Luminal ) [126]. We therefore mined the P. falciparum 3D7 proteome in PlasmoDB to shortlist proteins fulfilling both the criteria, i.e., (i) the presence of the RR8422 (Arg-Arg-Polar-Basic-Aliphatic-Aliphatic) motif (as a representation of the conserved RRRK motif commonly seen in CAMLs; [127]) within 50 amino acids from the N-terminus, and (ii) the presence of at least 3 TMDs (Fig 6A).…”

Looking for a safe haven: tail-anchored proteins and their membrane insertion pathways

“…Other than Get1/WRB, Get2/CAML has no sequence ortholog in plants. However, only recently, a functional Get2/CAML homolog has been identified in Arabidopsis using affinity purification-mass spectrometry ( Asseck et al, 2021 ). Despite low sequence similarity, the overall structure comprising three TMDs and a cytosolic N-terminal stretch of basic amino acid residues seem to be evolutionarily conserved to maintain a common function ( Asseck et al, 2021 ).…”

“…However, only recently, a functional Get2/CAML homolog has been identified in Arabidopsis using affinity purification-mass spectrometry ( Asseck et al, 2021 ). Despite low sequence similarity, the overall structure comprising three TMDs and a cytosolic N-terminal stretch of basic amino acid residues seem to be evolutionarily conserved to maintain a common function ( Asseck et al, 2021 ). Position-specific iterative- basic local alignment search tool (BLAST) analysis of the human CAML sequence revealed co-selection of the two functional domains, allowing the identification of orthologous genes also in distant phyla ( Borgese, 2020 ; Asseck et al, 2021 ).…”