Publication Date

5-2015

Advisor(s)

Donald B. Oliver

Department

Molecular Biology and Biochemistry

Language

English

Abstract

Approximately 30% of proteins produced in E. coli are translocated through the universally conserved SecYEG channel, either for co-translational insertion into the cytoplasmic membrane, or post-translational secretion into the periplasm or outer membrane. The oligomerization state of the active SecYEG complex is unknown and controversial, as many studies support either a monomer, or a back-to-back or front-to-front dimer, depending on the conditions of the experiment. A recent study by Park et al. (2012) employing an OmpA-GFP chimera to jam SecYEG at an intermediate stage of translocation to simulate ongoing protein transport shows that active translocation conditions favor the SecYEG monomer. However, this study measured levels of dimerization by disulfide cross-linking, which is quite specific and may be affected by conformational changes associated with translocation, thereby losing its ability to detect dimers. Utilizing this OmpA-GFP chimera and the more promiscuous cross-linker parabenzoylphenylalanine genetically engineered at the dimer interface, I observed a steady level of the back-to-back dimer of SecYEG during jammed or normal protein translocation conditions. I also created a SecY mutant at amino acid residue 20 of the back-to-back interface that possesses stronger cross-linking capability than previously observed in the literature. Utilizing this mutant I further demonstrated that SecYEG channels cleared of substrate by the translation initiation inhibitor kasugamycin also showed a similar level of dimer compared to untreated cells. Thus, the inactive state of the translocon is not predominantly populated by dimers, as the model that the monomer is the active form of the channel might predict. My results therefore show that the back-to-back dimer is unperturbed by the functional status of the SecYEG channel, implying that this dimer conformation is a stable physiological state of SecYEG that is neither favored nor disfavored by active translocation conditions.

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