Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion

Abstract

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

Mg2+-binding shifts the IM30 activity from membrane protection to membrane destabilization

“…As can be seen in Figure 3A, binding of IM30 to the SLB caused a shift of the resonance frequency, associated with increased damping of the resonance oscillation in the absence of Mg 2+ . The frequency shift reached a constant level at about -80 Hz after ~2500 s, as already shown previously (15). In stark contrast, in presence of Mg 2+ the frequency signal changes were only ~25% of the signal change observed in the absence of Mg 2+ (Fig.…”

“…To quantitatively analyze the impact of Mg 2+ on IM30 membrane binding , we next used a quartzcrystal microbalance (QCM) to follow the binding of IM30 to a solid supported lipid bilayer (SLB) (20% DOPG / 80% DOPC), as previously described in the absence of Mg 2+ (15). In QCM experiments, the resonance frequency of a quartz crystal is determined.…”

“…However, a fraction of the protein is attached to the chloroplasts' inner envelope or the cyanobacterial cytoplasmic membrane, as well as to thylakoid membranes (TMs) (4)(5)(6)(7). The activity of IM30 is broadly linked to the biogenesis/maintenance of TMs (recently reviewed in (8)), and two specific activities are currently studied and discussed in greater detail: (i) membrane protection (9)(10)(11)(12)(13)(14)(15) and (ii) membrane remodeling and repair (10,15,16). Protection and maintenance of the TM system are necessary to prevent energy dissipation and to maintain a proton gradient across the TM, a membrane system highly vulnerable to membrane damage (17).…”

“…IM30 appears to share its membranestabilizing and/or protecting activity with other proteins of the PspA/IM30 family, namely PspA and LiaH (reviewed in (18)). Membrane protection via IM30 and related proteins likely involves the formation of a protecting protein carpet on a membrane surface (19) to block proton leakage of damaged membranes (15). Remodeling of the internal TM system, on the other hand, is unique to cyanobacteria and chloroplasts where it is essential to adapt the size and structure of the TM system during TM development and in response to changing light conditions (20)(21)(22)).…”

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

“…VIPP1 rings and rods may serve a similar function for thylakoids. Interestingly, VIPP1 membrane binding was recently observed to be enhanced by acidic conditions in vitro (Siebenaller et al, 2020), implying that VIPP1 may be able to sense H + leakage in stressed thylakoid regions. However, it remains to be tested whether swollen thylakoids in the F4E and V11E mutants leak enough H + to influence luminal pH.…”