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

Abstract

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.

Di-arginine and FFAT-like motifs retain a subpopulation of PRA1 at ER-mitochondria membrane contact sites

“…The di-arginine motif is known to facilitate ER localization by recruiting the COPI coat [ 48 ]. We have previously completed an unbiased split-ubiquitin yeast two-hybrid screen to identify novel PRA1 binding partners and better understand its function in-vivo [ 22 ]. Among the new binding partners we uncovered and confirmed in mammalian cells was ζ1-COP, a member of the heptameric COPI coat [ 49 ].…”

“…Furthermore, in-vivo evidence points to a more structural role within the early secretory pathway as knock-down or knock-out of PRA1 leads to abnormal ER and Golgi phenotypes [ 18 – 21 ]. We recently used an unbiased approach that takes into account the membrane association of PRA1 to screen for novel binding partners and failed to identify any Rab GTPases [ 22 ]. Together, this suggests that PRA1 may have other roles that have not been elucidated.…”

Di-arginine and FFAT-like motifs retain a subpopulation of PRA1 at ER-mitochondria membrane contact sites

“…The di-arginine motif is known to facilitate ER localization by recruiting the COPI coat (Yuan et al, 2003). We have previously completed an unbiased split-ubiquitin yeast two-hybrid screen to identify novel PRA1 binding partners and better understand its function in-vivo (Abu Irqeba and Ogilvie, 2019). Among the new binding partners we uncovered and confirmed in mammalian 1-COP, a member of the heptameric COPI coat (Kuge et al, 1993).…”

“…Pulldown of PRA1 from both mammalian cells and yeast leads to co-immunoprecipitation of mostly ER localized proteins and fails to show that PRA1 associates with Rab GTPases (Geng et al, 2005; Huttlin et al, 2015). Our split-ubiquitin yeast two-hybrid screen also failed to show that PRA1 interacts with Rab GTPases (Abu Irqeba and Ogilvie, 2019).…”

“…Furthermore, in-vivo evidence points to a more structural role within the early secretory pathway as knock-down or knock-out of PRA1 leads to abnormal ER and Golgi phenotypes (Geng et al, 2005; Lee et al, 2017; Liu et al, 2011; Simpson et al, 2012). We recently used an unbiased approach that takes into account the membrane association of PRA1 to screen for novel binding partners and failed to identify any Rab GTPases (Abu Irqeba and Ogilvie, 2019). Together, this suggests that PRA1 may have other roles that have not been elucidated.…”