Open Collections

Identification of membrane-interacting proteins and membrane protein interactomes using Nanodiscs and proteomics

University of British Columbia

The insoluble nature of membrane proteins has complicated the identification of their interactomes. The Nanodisc has allowed the membrane and membrane proteins to exist in a soluble state. In this thesis, we combined Nanodisc and proteomics and applied the technique to discover the interactome of membrane proteins. Using the SecYEG and MalFGK membrane complex incorporated into Nanodisc, we identified, Syd, SecA, and MalE. These interactions were identified with high specificity and confidence from total soluble protein extracts. The protein YidC was also tested but no interactors were detected. Overall, these results showed that the technique can identify periplasmic and cytosolic interacting partners with high degree of specificity. In a second approach, the method was applied to detect proteins with high affinity for lipid using S. cerevisiae as a model organism. Using Nanodiscs containing different types of phospholipids, many known lipid interactors were identified, including: Ypt1, Sec4, Vps21, Osh6, and Faa1. Interestingly, Caj1 was identified as a PA specific interactor and this interaction was found to be pH dependent. Liposome sedimentation assay showed that Caj1 has affinity for acidic phospholipids. In vivo analysis confirmed the plasma membrane localization of N’-GFP-Caj1 and specifically to the yeast buds. However, pH dependent localization was not observed. Together, with the in vivo and in vitro results suggests that Caj1 is an acidic phospholipid interacting protein.

Text

2011-11-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/39413

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/