Abstract
In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.
Highlights
VIPP1 is a highly conserved protein found in cyanobacteria and the chloroplasts of both algae and land plants
VIPP1 binds to liposomes containing the anionic lipids phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) but not to liposomes composed of only neutral lipids[27]
To test whether this is true for chloroplast VIPP1, we recombinantly produced VIPP1 from Chlamydomonas reinhardtii
Summary
Chlamydomonas VIPP1 binds strongly to phosphatidylinositol phosphates. Cyanobacterial. As the VIPP1 rods were preassembled prior to liposome addition, the liposomes were likely drawn into the rods by a strong interaction with the rod’s inner surface until the entirety of the rod was filled, leaving the remainder of the liposome attached as a “bubble” on one end of the rod These cryo-ET observations strongly suggest that the larger size of VIPP1-containing particles upon incubation with PC:PI4P liposomes (Fig. 1) is due to the engulfment of the liposomes. The comparison between empty and liposome-encapsulating rods must be made within a single VIPP1 prep We performed this same-preparation comparison by negative stain and found that the addition of PC:PI4P liposomes only had a minor effect on VIPP1 rod diameter (Fig. 2d). To obtain the bending free energy per lipid molecule Gbliepnd, Eq 1 was divided by the number of lipid molecules in the cylinder nlip according to
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