Abstract
Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs.
Highlights
B. subtilis YidC1 requires the strictly conserved arginine for function
Arginine 366 in the E. coli YidC Is Not Required for Function—The crystal structure of B. halodurans YidC2 revealed a hydrophilic groove that was exposed to the cytoplasm and the inner bilayer leaflet (Fig. 1A) [21]
An arginine was present within the groove, and this arginine in the B. subtilis SpoIIIJ (YidC1) is essential for membrane protein insertion of the single spanning MifM protein. This positive charge is invariant in all YidC homologs in bacteria, mitochondria, and chloroplasts, even though little of the overall YidC sequence is conserved between the various organisms [11, 12], which suggests the positively charged residue is important for YidC function
Summary
B. subtilis YidC1 requires the strictly conserved arginine for function. Results: The conserved positively charged residue is critical for S. mutans YidC2 function but not for E. coli or chloroplast homologs. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. The conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. The conserved arginine is crucial for Bacillus subtilis SpoIIIJ (YidC1) to function, and SpoIIIJ was only able to rescue the growth of a SpoIIIJ depletion strain when the conserved arginine was changed to a lysine but not when mutated to a neutral or negatively charged residue This suggests that the positively charged residue is essential for YidC1 to facilitate membrane protein insertion in B. subtilis. The results described above indicate a more general role for this cytoplasmic hairpin domain in membrane protein insertion
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