2022 PRINCIPLES GUIDING IM30-MEDIATED MEMBRANE REMODELING

Authors:
Carmen Siebenaller

Journal:
Dissertation

Institute:

am Fachbereich Chemie, Pharmazie, Geographie und Geowissenschaften der Johannes Gutenberg-Universität Mainz

Abstract:

The inner membrane-associated protein of 30 kDa (IM30) is critically involved in the biogenesis and dynamics of thylakoid membranes (TMs). However, its exact physiological function is unclear so far. IM30 appears to be involved in both the stabilization as well as the continuous reorganization of the TM network. Membrane remodeling could involve the membrane-fusogenic activity of IM30 which is observed when Mg2+ is present. Since membrane fusion requires at least partial destabilization of the lipid bilayer, the two main functions of IM30 appear to be contradictory. Consequently, it is essential to clearly regulate these two functions in vivo. Since regulation of the IM30 activity is essentially not understood thus far, my research has focused on the impact of various factors on the in vitro structure and membrane interactions of IM30 of the cyanobacterium Synechocystis sp. PCC 6803.

My observations indicate that binding of nucleotides to IM30 and their hydrolysis do not affect oligomerization of the IM30 monomers. The results further suggest that the IM30 NTPase activity has no decisive impact on membrane interactions of IM30. Consequently, the relevance of nucleotide hydrolysis by IM30 remains to be shown. In contrast, mildly acidic pH (~ 5) had considerable effects on the IM30 structure, accompanied by an enhanced membrane binding propensity and membrane fusion. Since low pH likely occurs at defective TM regions where protons leak out of the TM lumen, IM30 is suggested to sense and seal proton leaks of damaged TM regions. Further observations indicate that IM30 rings disassemble on a membrane in absence of Mg2+, and that smaller IM30 oligomers have an increased membrane binding affinity. In contrast, increased binding of IM30 to both native and model membranes was observed in the presence of Mg2+, accompanied by IM30-induced membrane defects. Thus, Mg2+ might be a switch regulating the dual function of IM30. Another regulatory mechanism could be the phosphorylation of specific amino acids, since phosphomimetic mutations were shown to result in a decreased membrane fusion activity of IM30. Similar consequences were observed when the conserved Trp71 was mutated. Furthermore, a relevance of the TM-specific lipid MGDG for the IM30-mediated membrane fusion was revealed, since MGDG-containing membranes are considerably more resistant at high Mg2+-concentrations against induced membrane fusion.

In summary, the results of this thesis provide insights into the potential in vivo regulation of IM30 and suggest a structural basis of the dual IM30 function