Biochemical and structural insights into Rab12 interactions with RILP and its family members

Jana Omar,Yuto Maruta,Adva Yeheskel,Ronit Sagi-Eisenberg,Mitsunori Fukuda,Adi Efergan,Efrat Rosenbaum,Bayan Abu Sneineh

doi:10.1038/s41598-021-89394-y

Abstract

Alongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

Highlights

A screen of Rab GTPases for their functional and phenotypic impact on mast cell (MC) exocytosis has identified 30 Rabs as potential regulators of this p rocess[1]
We examined the capacity of GFP-fused versions of RILP, RILP-like 1 (RILP-L1) and RILP-like 2 (RILP-L2) to co-immunoprecipitate T7-tagged versions of themselves or the other members of this family, which we co-transfected in RBL cells, our model M Cs2
They demonstrated the ability of GFP-RILP-L1 to co-immunoprecipitate T7-RILP-L1, and that of GFP-RILP-L2 to coimmunoprecipitate T7-RILP-L2 (Fig. 1a)

Summary

Introduction

A screen of Rab GTPases for their functional and phenotypic impact on mast cell (MC) exocytosis has identified 30 Rabs as potential regulators of this p rocess[1]. Among these Rabs, a constitutively active mutant of Rab[12] was found to inhibit exocytosis by stimulating microtubule dependent retrograde transport of the MC secretory granules (SGs), promoting their perinuclear c lustering[1,2]. Rab GTPases perform their functions by the recruitment of effector proteins that bind to their active, GTP-bound conformation The latter include motor proteins, SNAREs, tethering factors, cytoskeleton and cargo proteins, whose recruitment allow Rabs to regulate distinct steps along vesicular trafficking[7].

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Results

Conclusion