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In search of tail-anchored protein machinery in plants: reevaluating the role of arsenite transporters

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ABSTRACT

Although the mechanisms underlying selective targeting of tail-anchored (TA) membrane proteins are well established in mammalian and yeast cells, little is known about their role in mediating intracellular membrane trafficking in plant cells. However, a recent study suggested that, in green algae, arsenite transporters located in the cytosol (ArsA1 and ArsA2) control the insertion of TA proteins into the membrane-bound organelles. In the present work, we overproduced and purified these hydrophilic proteins to near homogeneity. The analysis of their catalytic properties clearly demonstrates that C. reinhardtii ArsA proteins exhibit oxyanion-independent ATPase activity, as neither arsenite nor antimonite showed strong effects. Co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. Together with a structural model and molecular dynamics simulations, we propose that C. reinhadtii ArsA proteins are not arsenite transporters, but a TA-protein targeting factor. Further, we propose that ArsA targeting specificity is achieved at the ligand level, with ArsA1 mainly carrying TA-proteins to the chloroplast, while ArsA2 to the endoplasmic reticulum.

No MeSH data available.


Purifying Cr-ArsA2 and Cr-ArsA1.(a) Soluble, high level expression of Cr-ArsA2 in E. coli and purification by Ni-NTA chromatography. (b) Fractions from preparative size exclusion chromatography. (c) Expression of GST-Cr-ArsA1 in E. coli and purification by Ni-NTA chromatography. (d) Cr-ArsA1 was fractionated by size exclusion chromatography after the cleavage with 6xHis-tagged TEV protease and removal of residual uncleaved GST protein and 6xHis-tagged TEV protease by subtractive Ni-NTA purification. P, pellet; S, supernatant; E, elution.
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f2: Purifying Cr-ArsA2 and Cr-ArsA1.(a) Soluble, high level expression of Cr-ArsA2 in E. coli and purification by Ni-NTA chromatography. (b) Fractions from preparative size exclusion chromatography. (c) Expression of GST-Cr-ArsA1 in E. coli and purification by Ni-NTA chromatography. (d) Cr-ArsA1 was fractionated by size exclusion chromatography after the cleavage with 6xHis-tagged TEV protease and removal of residual uncleaved GST protein and 6xHis-tagged TEV protease by subtractive Ni-NTA purification. P, pellet; S, supernatant; E, elution.

Mentions: Cr-ArsA1 comprises a single polypeptide with two ATPase domains (~30% sequence identity to mammalian TRC40), whereas Cr-ArsA2 has only one ATPase domain with 40~50% sequence identity to TRC40 and Get3 (Fig. 1). A sequence alignment also reveals that both proteins contain three highly conserved, and essential regions for TRC40-like proteins, i.e. the P-loop, the switch I, and the switch II motifs18. Eukaryotic TRC40 enzymes also contain an approximately 20~30-residue insertion in the α-helical domain (TRC40-insert)18, which is absent from bacterial ArsA homologs, but found in Cr-ArsA2 as well as Cr-ArsA1 (Fig. 1). Both Cr-ArsA2 and Cr-ArsA1 lack conserved cysteine residues (Cys-113, Cys-172 and Cys-422 in E. coli ArsA) used by bacterial ArsA to bind metalloids35, and also lack the CXXC dimerization motif, generally present in eukaryotic TRC40/Get3. However, when recombinant Cr-ArsA2 was purified (Fig. 2), it formed both dimers (~80 kDa peak) and tetramers (~160 kDa peak, Fig. 2b). This is consistent with a previous study in the characterization of an archaeal Get3, which concluded that certain putative Get3 homologues may still oligomerize, even in the absence of CXXC motif24. As predicted by the alignment, purified Cr-ArsA1 revealed a monomeric architecture, with a molecular weight of about 80 kDa, much like bacterial ArsA proteins (Fig. 2c and d).


In search of tail-anchored protein machinery in plants: reevaluating the role of arsenite transporters
Purifying Cr-ArsA2 and Cr-ArsA1.(a) Soluble, high level expression of Cr-ArsA2 in E. coli and purification by Ni-NTA chromatography. (b) Fractions from preparative size exclusion chromatography. (c) Expression of GST-Cr-ArsA1 in E. coli and purification by Ni-NTA chromatography. (d) Cr-ArsA1 was fractionated by size exclusion chromatography after the cleavage with 6xHis-tagged TEV protease and removal of residual uncleaved GST protein and 6xHis-tagged TEV protease by subtractive Ni-NTA purification. P, pellet; S, supernatant; E, elution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382584&req=5

f2: Purifying Cr-ArsA2 and Cr-ArsA1.(a) Soluble, high level expression of Cr-ArsA2 in E. coli and purification by Ni-NTA chromatography. (b) Fractions from preparative size exclusion chromatography. (c) Expression of GST-Cr-ArsA1 in E. coli and purification by Ni-NTA chromatography. (d) Cr-ArsA1 was fractionated by size exclusion chromatography after the cleavage with 6xHis-tagged TEV protease and removal of residual uncleaved GST protein and 6xHis-tagged TEV protease by subtractive Ni-NTA purification. P, pellet; S, supernatant; E, elution.
Mentions: Cr-ArsA1 comprises a single polypeptide with two ATPase domains (~30% sequence identity to mammalian TRC40), whereas Cr-ArsA2 has only one ATPase domain with 40~50% sequence identity to TRC40 and Get3 (Fig. 1). A sequence alignment also reveals that both proteins contain three highly conserved, and essential regions for TRC40-like proteins, i.e. the P-loop, the switch I, and the switch II motifs18. Eukaryotic TRC40 enzymes also contain an approximately 20~30-residue insertion in the α-helical domain (TRC40-insert)18, which is absent from bacterial ArsA homologs, but found in Cr-ArsA2 as well as Cr-ArsA1 (Fig. 1). Both Cr-ArsA2 and Cr-ArsA1 lack conserved cysteine residues (Cys-113, Cys-172 and Cys-422 in E. coli ArsA) used by bacterial ArsA to bind metalloids35, and also lack the CXXC dimerization motif, generally present in eukaryotic TRC40/Get3. However, when recombinant Cr-ArsA2 was purified (Fig. 2), it formed both dimers (~80 kDa peak) and tetramers (~160 kDa peak, Fig. 2b). This is consistent with a previous study in the characterization of an archaeal Get3, which concluded that certain putative Get3 homologues may still oligomerize, even in the absence of CXXC motif24. As predicted by the alignment, purified Cr-ArsA1 revealed a monomeric architecture, with a molecular weight of about 80 kDa, much like bacterial ArsA proteins (Fig. 2c and d).

View Article: PubMed Central - PubMed

ABSTRACT

Although the mechanisms underlying selective targeting of tail-anchored (TA) membrane proteins are well established in mammalian and yeast cells, little is known about their role in mediating intracellular membrane trafficking in plant cells. However, a recent study suggested that, in green algae, arsenite transporters located in the cytosol (ArsA1 and ArsA2) control the insertion of TA proteins into the membrane-bound organelles. In the present work, we overproduced and purified these hydrophilic proteins to near homogeneity. The analysis of their catalytic properties clearly demonstrates that C. reinhardtii ArsA proteins exhibit oxyanion-independent ATPase activity, as neither arsenite nor antimonite showed strong effects. Co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. Together with a structural model and molecular dynamics simulations, we propose that C. reinhadtii ArsA proteins are not arsenite transporters, but a TA-protein targeting factor. Further, we propose that ArsA targeting specificity is achieved at the ligand level, with ArsA1 mainly carrying TA-proteins to the chloroplast, while ArsA2 to the endoplasmic reticulum.

No MeSH data available.