PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

The IM30/Vipp1 C-terminus associates with the lipid bilayer and modulates membrane fusion.

IM30/Vipp1 proteins are crucial for thylakoid membrane biogenesis in chloroplasts and cyanobacteria. A characteristic C-terminal extension distinguishes these proteins from the homologous bacterial PspA proteins, and this extension has been discussed to be key for the IM30/Vipp1 activity. Here we report that the extension of the Synechocystis IM30 protein is indispensable, and argue that both, the N-terminal PspA-domain as well as the C-terminal extension are needed in order for the IM30 protein to conduct its in vivo function. In vitro, we show that the PspA-domain of IM30 is vital for stability/folding and oligomer formation of IM30 as well as for IM30-triggered membrane fusion. In contrast, the IM30 C-terminal domain is involved in and necessary to stabilize defined contacts to negatively charged membrane surfaces, and to modulate the IM30-induced membrane fusion activity. Although the two IM30 protein domains have distinct functional roles, only together they enable IM30 to work properly.

Organization into Higher Ordered Ring Structures Counteracts Membrane Binding of IM30, a Protein Associated with Inner Membranes in Chloroplasts and Cyanobacteria.

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.

Structural and functional analysis of the archaeal endonuclease Nob1.

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

Vipp1: a very important protein in plastids?!

As a key feature in oxygenic photosynthesis, thylakoid membranes play an essential role in the physiology of plants, algae, and cyanobacteria. Despite their importance in the process of oxygenic photosynthesis, their biogenesis has remained a mystery to the present day. A decade ago, vesicle-inducing protein in plastids 1 (Vipp1) was described to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Most follow-up studies clearly linked Vipp1 to membranes and Vipp1 interactions as well as the defects observed after Vipp1 depletion in chloroplasts and cyanobacteria indicate that Vipp1 directly binds to membranes, locally stabilizes bilayer structures, and thereby retains membrane integrity. Here current knowledge about the structure and function of Vipp1 is summarized with a special focus on its relationship to the bacterial phage shock protein A (PspA), as both proteins share a common origin and appear to have retained many similarities in structure and function.

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

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.

IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

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+), 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.