Publication Date

5-2018

Advisor(s)

Donald B. Oliver

Department

Molecular Biology & Biochemistry

Abstract

SecYEG is the core translocon in the Sec pathway that is essential for the transport of most of the secretome in the three kingdoms of life. Understanding whether it works as a monomer or a dimer is central to the elucidating how this pathway works. Chapter II of my thesis focuses on deciphering the oligomeric state of SecYEG during protein translocation. This work mainly employed in vivo photo-crosslinking to determine the oligomeric state of SecYEG during both translocating and resting states. Our results indicate that the SecYEG monomer to dimer ratio is refractory to the global translocon jamming induced by the substrate OmpA-GFP. This result was observed for both the front-to-front and back-to-back dimer states. The OmpA-GFP-SecY immunoprecipitation and SecA foot-printing studies also indicate that SecY dimer is involved in active protein translocation. The latter study specifically supports the in SecYEG is the core translocon in the Sec pathway that is essential for the transport of most of the secretome in the three kingdoms of life. Understanding whether it works as a monomer or a dimer is central to the elucidating how this pathway works. Chapter II of my thesis focuses on deciphering the oligomeric state of SecYEG during protein translocation. This work mainly employed in vivo photo-crosslinking to determine the oligomeric state of SecYEG during both translocating and resting states. Our results indicate that the SecYEG monomer to dimer ratio is refractory to the global translocon jamming induced by the substrate OmpA-GFP. This result was observed for both the front-to-front and back-to-back dimer states. The OmpA-GFP-SecY immunoprecipitation and SecA foot-printing studies also indicate that SecY dimer is involved in active protein translocation. The latter study specifically supports the in vivo translocation activity of the back-to-back dimer. Collectively, our results contradict an earlier study by Park et al. which claimed that SecYEG dimer dissociates during protein translocation. The apparent cause for the different results obtained in the parallel portions of our two studies is unclear.

Chapter III of my thesis is a continuation of my work in chapter II. Although we obtained evidence that SecYEG dimer is involved in protein translocation, we couldn't tell whether dimer initiates translocation or remains in this states through-out the entire reaction, or whether there is room for monomer function at some portion of the translocation cycle. The focus of chapter III is to determine the function of the SecYEG monomer. We created a previously described secYE double mutant that according to disulfide crosslinking was defective in the formation of both the front-to-front and back-to-back dimers, and thus presumably to produce only monomers. We explored the function of such a mutant and compared it to the wildtype strain by investigating the translocation efficiency of secretory proteins including OmpA, MBP, GBP, and an integral membrane chimeric protein MalF-PSBT, whose membrane insertion could be monitored by its post-translational modification by cytoplasmic biotin ligase. Our results indicated that the secYE mutant has a comparable protein transport activity as the isogenic wildtype strain when the mutant strain was corrected for a modest defect in SecYEG assembly rate. Since the mutant was essentially normal in its protein transport activity, we decided to readdress the issue as to whether its translocon was indeed solely in a monomeric state or not using two alternative methods of assessment. The subsequent SecA-foot-printing and in vivo photo-crosslinking studies showed that the mutant can form dimers in both the translocating and the resting state at similar levels to the wildtype. We conclude that more work will be needed to generate a strain that truly produces solely a SecYEG monomer. Alternative strategies for the study of the function of the monomer are also suggested in the Discussion chapter.

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