A Janus-Faced IM30 Ring Involved in Thylakoid Membrane Fusion Is Assembled from IM30 Tetramers

Abstract

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.

Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding

“…140 Å (i.e. a double ring has a height of ϳ280 Å) (16), solely formation of double rings via the ring top surfaces containing the C termini (that carry CFP/YFP) satisfactorily explains the observed increase in the FRET signals upon Mg 2ϩ -induced double ring formation (Fig. 5A).…”

“…Thus, increasing the Mg 2ϩ availability in the stroma/cytosol on one hand might result in an increased membrane-binding affinity of soluble IM30 rings because of the increased hydrophobicity of the protein surface, but could also alter the structure of membrane-attached IM30 rings. Membrane fusion involves the ability to connect two membrane layers, and thus Mg 2ϩ activation of IM30 rings has to involve formation of IM30 rings exposing hydrophobic regions at both the ring top and bottom (16). Although…”

“…However, whereas PspA appears to form one type of ring with a well-defined size and a 9-fold internal symmetry (13), IM30 forms diverse oligomeric rings with varying rotational symmetries of at least 7-22 (11,12). We were recently able to generate a 3D surface representation of several IM30 rings, and based on these structures and mutant analyses we could show that the ring top and bottom clearly differ, and the IM30-specific C terminus, for example, is located exclusively at the ring top side (16). However, because a detailed structure of full-length IM30 or PspA is not available yet, it is still unclear how the monomers precisely pack inside these rings.…”

What is Vipp1 good for?

“…It has been calculated that the formed puncta contain ∼ 100 to 300 GFP molecules and exhibit an estimated diameter of ∼ 100 nm (Bryan et al ., ). When free in solution, Vipp1/IM30 forms oligomeric rings, consisting of (at least) 22–88 monomers with diameters of 24–32 nm and a height of ∼ 15 nm (Fuhrmann et al ., ; Saur et al ., ). Thus, based on the observed dimensions of the localized puncta, these could be formed by several rings (Bryan et al ., ).…”

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression

“…In both PspA and VIPP1, the N‐terminal 24 amino acids form an amphipathic α‐helix (AHa) that is required for membrane binding and the formation of larger oligomers (Gao, Wang, Zhang, & Liu, ; Jovanovic et al, ; McDonald, Jovanovic, Ces, & Buck, ; McDonald, Jovanovic, Wallace, Ces, & Buck, ; Otters et al, ). In vitro, both proteins form higher order oligomers of >1 MDa that give rise to rings and rods (Aseeva et al, ; Fuhrmann, Bultema, et al, ; Hankamer, Elderkin, Buck, & Nield, ; Liu et al, ; Male, Oyston, & Tavassoli, ; Saur et al, ). Rods formed by recombinant VIPP1 from Chlamydomonas can engulf liposomes containing physiological amounts of phosphatidylinositol phosphate (Theis, Gupta, et al, ).…”