Poster abstracts

Poster number 48 submitted by Lu Zhu

YidC is important for folding of LacY into its tertiary conformation and responding of LacY to ligand binding

Lu Zhu (The Ohio State University, Department of Chemistry, Columbus, OH 43210), H. Ronald Kaback (Department of Physiology, University of California Los Angeles, Los Angeles, California, USA.), Ross Dalbey (The Ohio State University, Department of Chemistry, Columbus, OH 43210)

Abstract:
YidC, an evolutionarily conserved protein, plays an important role in the biogenesis of membrane protein in bacteria. Here, we use the model inner membrane protein LacY to study how YidC depletion impairs the insertion and folding of a polypotic membrane. With a well-established indirect chemical shift assay, we find that LacY is inserted into inner membrane through a Sec dependent and SRP dependent pathway. Moreover, YidC does not play as an insertase for LacY in that all six periplasmic loops of LacY are shown to be fully translocated into inner membrane in the YidC depleted condition. The findings are consistent with previous protease mapping data of LacY. [Shushi N., Ronald Kaback., et al. (2004) JCB vol. 165 no. 1 53-62] Interestingly, YidC can be an insertase for a hybrid polytopic membrane protein, LacY-Procoat (N6-M13 procoat-C6). By using site-directed chemical crosslinking, we show that helix proximity between some transmembrane segments and ligand-induced conformational changes in the LacY were perturbed by YidC depletion. We also use membrane permeable thiol reagent N-ethyl-ethylmaleimide (NEM) to detect site-directed alkylation of engineered single-Cys mutants with or without ligand in situ. Remarkably, the solvent accessibility of some periplasmic surface Cys residues and increased accessibility of Cys residues to the aqueous environment in the periplasmic region of the helice are changed upon the YidC depletion. The results confirm and support that YidC plays as a primary role in folding of LacY into its tertiary conformation and responding of LacY to ligand binding.

References:
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Keywords: LacY, YidC, membrane protein