Novel binding partners for Prenylated Rab Acceptor 1 identified by a split-ubiquitin yeast two-hybrid screen.

Authors:
Judith Mosinger Ogilvie
Judith Mosinger Ogilvie
Saint Louis University
United States

BMC Res Notes 2019 Mar 29;12(1):188. Epub 2019 Mar 29.

Biology Department, Saint Louis University, Macelwane Hall, 3507 Laclede Ave, St. Louis, MO, 63103, USA.

Objective: Prenylated Rab Acceptor 1 (PRA1) is a transmembrane protein localized to the early secretory pathway. It has been found to interact with an array of Rab GTPases, leading to its hypothesized function in the recycling of Rab GTPases. However, all previous strategies used to screen for novel interacting partners have utilized a classic yeast two-hybrid approach that requires both bait and its potential binding partners to be cytosolic proteins. In the split-ubiquitin yeast two-hybrid screen, a protein interaction leads to the re-constitution of ubiquitin, which is followed by proteolytic release of a transcription activator that migrates to the nucleus alone. This allows for bait and/or prey to be integral membrane protein(s). To better understand the in vivo function of PRA1, we took an unbiased approach that screened PRA1 against a normalized mouse neuronal cDNA library using this variant of the classic screening strategy.

Results: We report 41 previously unidentified potential PRA1 binding partners revealed by this screen and validate the screen by confirming three of these interactions using a bi-molecular fluorescence complementation assay in mammalian cells. The identified proteins reside throughout the secretory pathway and are both membrane-bound and cytosolic in their identity, suggesting alternative functions for PRA1.

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Source
http://dx.doi.org/10.1186/s13104-019-4219-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441142PMC
March 2019

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