Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Małgorzata Gutkowska,Magdalena Wojtas,Michał Dadlez,Małgorzata Lichocka,Marta Hoffman‐Sommer,Magdalena Małgorzata Pamuła,Anna Daria Leja,Agnieszka Witek,Ewa Swiezewska,Magdalena Kaus‐Drobek,Liliana Surmacz,Małgorzata Perycz

doi:10.1111/tpj.15519

Abstract

SUMMARYLipid anchors are common post‐translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C‐termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP–Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to describe the structural basis of plant REP–Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule‐specific, and in other cases they are common for a subset of Rabs. In particular, the C‐terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C‐terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

Highlights

Rab proteins belong to a large family of small GTPases – regulatory proteins important for vesicular transport and signal transduction in the cell (Pfeffer, 2017)
Recombinant plant Rab escort protein (REP) forms in vitro stoichiometric complexes with selected Rab proteins We were interested to find out which parts of the REP and Rab proteins are engaged in complex formation in plants
We wanted to know if the REP C-terminal tail takes part in the interaction and whether the same regions of REP were involved with different Rabs

Summary

Introduction

Rab proteins belong to a large family of small GTPases – regulatory proteins important for vesicular transport and signal transduction in the cell (Pfeffer, 2017). Specific Rab subclasses have undergone expansion in plants (e.g., the Rab-A family is expanded compared to Rab in mammals), while other subclasses are more expanded in mammals than in plants (e.g., the small Rab-E group in plants compared to the broad expansion of Rab8-related groups in mammals; Rutherford and Moore, 2002) This is likely connected to the fact that plants, being sessile organisms, have many additional requirements for specific functions that are absent or less developed in motile organisms. These functions may include the biosynthesis and modification of cell walls, defense against pathogens, phytohormone transport, or maintaining ion homeostasis and regulating vacuolar storage (Elliott et al, 2020; Nielsen, 2020; Rivero et al, 2019). The need for both constitutive and specialized traffic puts Rab proteins in a central

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