Abstract
The function of IM30 (also known as Vipp1) is linked to protection and/or remodeling of the thylakoid membrane system in chloroplasts and cyanobacteria. Recently, it has been revealed that the Arabidopsis IM30 protein exhibits GTP hydrolyzing activity in vitro, which was unexpected, as IM30 does not show any classical GTPase features. In the present study, we addressed the question, whether an apparent GTPase activity is conserved in IM30 proteins and can also be observed for IM30 of the cyanobacterium Synechocystis sp. PCC 6803. We show that Synechocystis IM30 is indeed able to bind and hydrolyze GTP followed by the release of Pi. Yet, the apparent GTPase activity of Synechocystis IM30 does not depend on Mg2+, which, together with the lack of classical GTPase features, renders IM30 an atypical GTPase. To elucidate the impact of this cryptic GTPase activity on the membrane remodeling activity of IM30, we tested whether GTP hydrolysis influences IM30 membrane binding and/or IM30-mediated membrane fusion. We show that membrane remodeling by Synechocystis IM30 is slightly affected by nucleotides. Yet, despite IM30 clearly catalyzing GTP hydrolysis, this does not seem to be vital for its membrane remodeling function.
Highlights
IM30, the inner membrane-associated protein of 30 kDa ( known as Vipp[1]), is present in cyanobacteria as well as in chloroplasts of higher plants and algae[1]
Our results suggest that the Synechocystis IM30 has a low GTP hydrolyzing activity, as observed before with the Arabidopsis homolog, yet nucleotides have only minor effects on the in vitro membrane remodeling activity of IM30, and nucleotide binding/hydrolysis does not appear to critically affect IM30-triggered membrane remodeling
We tested whether SynIM30 hydrolyzes GTP, using the malachite green-based assay that has been used by Ohnishi et al to determine the apparent GTPase activity of AraIM3039
Summary
IM30, the inner membrane-associated protein of 30 kDa ( known as Vipp[1] (vesicle-inducing protein in plastids 1)), is present in cyanobacteria as well as in chloroplasts of higher plants and algae[1]. While diverse functions have been attributed to IM30 in the past[1,2,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24], most observations clearly indicate that the protein is membrane-active and involved in the biogenesis, dynamics and/or stabilization of internal membranes in chloroplasts and cyanobacteria (recently reviewed in[25]). It was somewhat intriguing to suspect a nucleotide binding/hydrolysis function for IM30, a protein that can mediate membrane fusion[23] Consistent with this idea, Ohnishi et al recently suggested that recombinant Arabidopsis IM30 (AraIM30) has an intrinsic GTPase activity despite lacking classical features of GTPases, implying that IM30 belongs to a new class of membrane-remodeling GTPases[39]. Our results suggest that the Synechocystis IM30 has a low GTP hydrolyzing activity, as observed before with the Arabidopsis homolog, yet nucleotides have only minor effects on the in vitro membrane remodeling activity of IM30, and nucleotide binding/hydrolysis does not appear to critically affect IM30-triggered membrane remodeling
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