The Major Periplasmic Domain of YidC May Be Required for Polar Localization of a Green Fluorescence Protein Tagged YidC Variant Protein in Escherichia coli

Membrane proteins play an essential role in the survival of prokaryotic cells. YidC is a transmembrane protein that functions to insert proteins into the cell membrane either through the Sec translocase dependent pathway or an alternative independent pathway. YidC is 548 amino acids in length, and is comprised of six transmembrane domains and numerous cytoplasmic/periplasmic domains, the biggest being the first periplasmic domain (P1) which is 319 amino acids long. The specific function of YidC P1 is still not well characterized. Previous studies have shown that YidC fused to green fluorescence protein localizes to the cell poles. We hypothesize that the P1 periplasmic region directs the polar localization of YidC. Here we describe a PCR deletion method, using homologous-end designed primers to create a deletion of the P1 domain in a YidC-GFP variant. The resulting nucleotide sequence of the deletion construct was determined to confirm the in-frame deletion in P1 of YidC-GFP. Fluorescence and bright field microscopy were used to observe localization of the mutant YidC-GFP protein. BL21 (DE3) cells expressing either the YidC-GFP P1 deletion construct or the YidC-GFP wild type construct were compared. Our preliminary observations suggest that deletion of the YidC-GFP P1 domain results in circumferential localization of the YidC protein in BL21 cells whereas wild type YidC-GFP was observed at the cell poles. We also conclude the deletion of the P1 region in YidC-GFP does not affect cell viability.