The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg 2 þ , membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.

Biogenesis and dynamics of thylakoid membranes likely involves membrane fusion events. Membrane attachment of the inner membrane-associated protein of 30 kDa (IM30) affects the structure of the lipid bilayer, finally resulting in membrane fusion. Yet, how IM30 triggers membrane fusion is largely unclear. IM30 monomers pre-assemble into stable tetrameric building blocks, which further align to form oligomeric ring structures, and differently sized IM30 rings bind to membranes. Based on a 3D reconstruction of IM30 rings, we locate the IM30 loop 2 region at the bottom of the ring and show intact membrane binding but missing fusogenic activity of loop 2 mutants. However, helix 7, which has recently been shown to mediate membrane binding, was located at the oppossite, top side of IM30 rings. We propose that a two-sided IM30 ring complex connects two opposing membranes, finally resulting in membrane fusion. Thus, IM30-mediated membrane fusion requires a Janus-faced IM30 ring.

The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30.Engulfment of an ancient cyanobacterium by a primordial cell has resulted in the development of modern day chloroplasts, the organelles where photosynthesis takes place. Consequently, the fine structures of cyanobacterial cells and chloroplasts share many similarities. Notably, chloroplasts and cyanobacteria both contain an extra internal membrane system, the thylakoid membrane (TM) 2 network. Furthermore, both perform oxygenic photosynthesis, and the TMs harbor all protein complexes and pigments involved in the photosynthetic light reaction. However, although assembly and maintenance of TMs is vital for photosynthesis, the details of TM biogenesis and maintenance are still largely unsolved mysteries (1).In 1994 the IM30 (inner membrane-associated protein of 30 kDa) was discovered in chloroplasts of Pisum sativum (2), and a homologous protein appears to be expressed in almost every organism that is able to conduct oxygenic photosynthesis (2-5). Several in vivo studies have shown that the protein plays an essential role in TM formation and maintenance (reviewed in Ref. 6), e.g. the depletion of the protein in Arabidopsis thaliana or the cyanobacterium Synechocystis sp. PCC 6803 has resulted in a decreased amount of TMs (3, 7-9). Under cold stress conditions, depletion of IM30 resulted in the formation of vesicular structures at the chloroplast inner envelope membrane in Arabidopsis, which led to the name Vipp1 (vesicle inducing protein in plastids 1) (3). Nevertheless, although diverse physiological functions were proposed for IM30 in the past two decades, the exact protein function remained elusive for a long time. Only recently, IM30 has been identified to be able to reorganize lipid bilayer structures and to mediate membrane fusion in chloroplasts and cyanobacteria (10). Similar to its bacterial homolog, the PspA (phage shock protein A), which is involved in a bacterial stress response in situations provoking membrane stress (11), IM30 specifically interacts wi...

The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg However, the precise role of Mg in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg binds directly to IM30 with a binding affinity of ∼1 mm Mg binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation because of increased exposure of hydrophobic surface regions. However, Mg-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.