Poster abstracts
Poster number 71 submitted by Balasubramani Hariharan
Polarity and Charge as determinants for translocase requirement for Membrane protein insertion.
Balasubramani Hariharan (Biophysics Graduate Program), Raunak Soman (OSBP)
Abstract:
Membrane proteins make up to a third of the cellular proteome and are involved in several essential functions of a cell. Almost 60% of drugs targeted are against membrane proteins, making its biogenesis one of the most prominent area in the field of drug development. The two key components discovered so far in the process of membrane protein insertion into the inner membrane of bacteria are YidC translocase and the Sec translocon complex (SecYEG). Both proteins are essential, universally conserved and together insert about 95% of the proteins into the inner membrane. Membrane insertion of SecYEG-dependent proteins occurs at the lateral gate of SecY, the channel-forming subunit of the bacterial Sec translocon. YidC, on the other hand, whose crystal structure was recently solved showed the presence of a hydrophilic groove open to both the cytoplasm and the lipid bilayer. Structure guided biochemical assays unveiled a mechanism of insertion for single transmembrane (TM) proteins which involves a negatively charged residue in the N-terminus of the substrate interacting with a positively charged residue in the hydrophilic groove of YidC with the assistance of Sec translocon. We hypothesize that, for larger membrane proteins, the polarity and charge of the periplasmic regions determine the YidC and Sec translocase requirements for insertion. Here we have tested this hypothesis by showing that procoat-Lep insertion (2 TM protein) can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent substrates. We test whether the length of procoat-Lep loop and the positive charge in the loop are determinants for Sec dependence. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was previously not inserted by native TM segment. This study throws light on the requirements of both the insertase to coexist in all walks of life. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC.
Keywords: Membrane proteins, translocation, YidC