Figure 1.

Proteins of the Oxa1 family share common structural features. (A) Comparison of the crystal structure of YidC from Bacillus halodurans (left; PDB: 3WO6; Kumazaki et al., 2014a) and the AlphaFold model of Arabidopsis thaliana Alb3 without signal peptide (middle; model: AF-Q8LBP4-F1; Jumper et al., 2021). Regions in the structural model of Alb3 highlighted in dark blue indicate conserved sequence motifs involved in cpSRP43 binding (residues 314–318 within the fourth TMH and motifs II and IV in the C-terminal region); the conserved motifs I and III are also highlighted in dark blue. The red arrowheads indicate the alkaline phosphatase (AP) insertion sites mentioned in C. Right: scheme of the predicted topology of the hydrophobic core region of members of the Oxa1 family encompassing five transmembrane helices. (B) Features of the C-terminal sequences of members of the Oxa1 family. The start of the C-terminal regions were defined to the first residue after the last transmembrane helix according to the crystal structure of Escherichia coli YidC (PDB: 3WVF; Kumazaki et al., 2014b) and the Alphafold models of A. thaliana Alb3 (see above) and Saccharomyces cerevisiae Oxa1 (model: AF-P39952-F1; Jumper et al., 2021). Theoretical pI-values for the indicated C-terminal peptide sequences were calculated using ExPASy ProtParam. Prominent sequence motifs are marked. I, II, III, and IV: conserved motifs of the Alb3 C-terminal region (Falk et al., 2010); RBD: conserved helical ribosome binding domain of the Oxa1 C-terminal region (Jia et al., 2003). (C) Alkaline phosphatase (AP) activity of bacterial cells carrying expression plasmids coding for different Alb3-AP fusions to identify membrane protein topology. AP was fused into mature Alb3 at amino acid positions 186 (mAlb3-AP-186), 231 (mAlb3-AP-231), 304 (mAlb3-AP-304), and to the C-terminus (mAlb3-AP-C). Control cells expressed the precursor of AP (pAP), mature AP (mAP) and mAlb3. The positions of the fusions are indicated schematically in A (red arrowheads).