Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Sri Karthika Shanmugamthesis.pdf (2.27 MB)

ETD Abstract Container

Abstract Header

Investigation of amino-tail translocation by the conserved YidC, Sec and independent pathways

Shanmugam, Sri Karthika
http://rave.ohiolink.edu/etdc/view?acc_num=osu15464319086543

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Chapter 1 of this dissertation reviews existing knowledge of the areas of protein translocation and membrane insertion. The core machineries involved in membrane protein biogenesis are remarkably conserved. Proteins that fold within the cell prior to export are translocated by the Tat system. Canonical substrates of this pathway possess signal sequences with a twin-arginine motif which interacts with the TatABC membrane translocon complex to facilitate substrate translocation. The majority of the secreted and membrane proteins are translocated by the Sec machinery in an unfolded state. It consists of the SecY, SecE and SecG proteins which form an hour-glass shaped channel with a lateral gate opening into the membrane. Substrate targeting to the Sec translocon occurs either post-translationally or co-translationally. Most exported proteins in E. coli are post-translationally targeted by the SecA/B pathway. The SecB is a molecular chaperone that delivers a subset of substrate proteins in an unfolded state to SecA. The SecA motor ATPase powers the movement of substrates through the SecYEG channel. Co-translational targeting typically involves the association of the translating ribosome with the translocase directly, or with the translocase after delivery by the signal-recognition particle (SRP) and its receptor (SR). Certain substrates of the SRP pathway are targeted to another translocon, YidC. YidC plays a pivotal role in the membrane integration, folding and assembly of a subset of proteins including energy-transducing and respiratory complexes. It functions both autonomously and in concert with the SecYEG channel in bacteria. The YidC family of proteins are widely conserved in all domains of life with new members recently identified in the eukaryotic ER membrane. Bacterial and organellar members share the conserved 5 TM core which forms a unique hydrophilic cavity in the inner leaflet of the bilayer accessible from the cytoplasm and the lipid phase. The work presented here investigates the pathway-determining factors for amino-terminal translocation in E. coli. In addition, the conserved function of the YidC family of proteins to insert a single-spanning protein into the membrane was explored using biophysical methods. Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion and assembly of inner membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In Chapter 2, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36 residue N-tail of 2Pf3-Lep translocates independent of YidC-Sec. This is the longest N-tail region that is translocated by this pathway. We confirmed this using a newly constructed YidC-Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC-Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted, however an MBP-2Pf3-Lep fusion protein could be translocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC-Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in E. coli. The YidC family of proteins share structural homology and engage in the process of membrane protein biogenesis of various cellular and organellar membranes. Chapter 4 explores the functional conservation amongst the eukaryotic YidC homologs by testing the insertion of the YidC-only model substrate Pf3 coat protein. Fluorescence correlation spectroscopy technique was utilized to follow the insertion process at the single-molecule level in vitro. The thylakoid membrane of chloroplasts contains 2 YidC-paralogs: Alb3 and Alb4. Although both have distinct set of substrates in the chloroplasts, we found that they can insert Pf3 coat substrate with comparable efficiencies to YidC. This is in agreement with the fact that these insertases can complement YidC in E. coli. We also tested the mitochondrial homolog Oxa1 and the newly proposed ER-resident member Get1. Oxa1 is a bonafide member of the YidC family; both YidC and Oxa1 can complement one another. However, experimental evidence is lacking to confirm the placement of Get1 in the YidC family. Interestingly, both Oxa1 and Get1 can insert Pf3 coat protein into reconstituted proteoliposomes. This suggests that Get1 and the other homologs tested are functionally conserved. This study provides fundamental information about the evolutionarily conserved role of YidC in membrane protein biogenesis.
Ross Dalbey (Advisor)
Thomas Magliery (Committee Member)
Natacha Ruiz (Committee Member)
James Cowan (Committee Member)
125 p.
Biochemistry; Biophysics
YidC; Sec; Membrane insertion; Alb3; Alb4; Oxa1; Get1; pathway-selection; translocation

Recommended Citations

Citations

  • Shanmugam, S. K. (2019). Investigation of amino-tail translocation by the conserved YidC, Sec and independent pathways [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu15464319086543

    APA Style (7th edition)

  • Shanmugam, Sri Karthika. Investigation of amino-tail translocation by the conserved YidC, Sec and independent pathways. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu15464319086543.

    MLA Style (8th edition)

  • Shanmugam, Sri Karthika. "Investigation of amino-tail translocation by the conserved YidC, Sec and independent pathways." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu15464319086543

    Chicago Manual of Style (17th edition)

osu15464319086543
131
© 2019, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.