The thylakoid membrane remodeling protein VIPP1 forms bundled oligomers in tobacco chloroplasts.

The thylakoid membrane (TM) serves as the scaffold for oxygen-evolving photosynthesis, hosting the protein complexes responsible for the light reactions and ATP synthesis. Vesicle inducing protein in plastid 1 (VIPP1), a key protein in TM remodeling, has been recognized as essential for TM homeostasis. In vitro studies of cyanobacterial VIPP1 demonstrated its ability to form large homo-oligomers (2†MDa) manifesting as ring-like or filament-like assemblies associated with membranes. Similarly, VIPP1 in Chlamydomonas reinhardtii assembles into rods that encapsulate liposomes or into stacked spiral structures. However, the nature of VIPP1 assemblies in chloroplasts, particularly in Arabidopsis, remains uncharacterized. Here, we expressed Arabidopsis thaliana VIPP1 fused to GFP (AtVIPP1-GFP) in tobacco (Nicotiana tabacum) chloroplasts and performed transmission electron microscopy (TEM). A purified AtVIPP1-GFP fraction was enriched with long filamentous tubule-like structures. Detailed TEM observations of chloroplasts in fixed resin-embedded tissues identified VIPP1 assemblies in situ that appeared to colocalize with GFP fluorescence. Electron tomography demonstrated that the AtVIPP1 oligomers consisted of bundled filaments near membranes, some of which appeared connected to the TM or inner chloroplast envelope at their contact sites. The observed bundles were never detected in wild-type Arabidopsis but were observed in Arabidopsis vipp1 mutants expressing AtVIPP1-GFP. Taken together, we propose that the bundled filaments are the dominant AtVIPP1 oligomers that represent its static state in vivo.