Abstract
The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.
Highlights
The “inner membrane–associated protein of 30 kDa” (IM30), known as “vesicle-inducing protein in plastids 1” (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts
We have demonstrated that IM30 mediates membrane fusion in presence of Mg2ϩ [10]
The exact role of Mg2ϩ in the fusion process is still enigmatic, and either (i) Mg2ϩ binding to IM30 results in a rearrangement of the IM30 structure that, in the end, triggers membrane fusion or (ii) alternatively, Mg2ϩ serves as a bridging ion that, gluelike, connects IM30 with the membrane
Summary
We have demonstrated that IM30 mediates membrane fusion in presence of Mg2ϩ [10]. To further support the assumption that Mg2ϩ binding alters the IM30 structure, we measured ANS fluorescence at increasing Mg2ϩ concentrations, using chemically cross-linked IM30 rings (IM30-X). All results obtained far clearly indicate that Mg2ϩ binding induces structural rearrangements, and the proteolysis data suggest a different structure and/or position of the IM30 C terminus in presence of Mg2ϩ. TEM of negatively stained IM30 indicated an increased formation of IM30 double rings and a small number of rod structures in presence of Mg2ϩ (Fig. 5C) Such aggregates can be observed in absence of Mg2ϩ, but to a much lower extent [11]. The structural rearrangements observed upon addition of Mg2ϩ result in ring stacking, which leads to an overall shielding of the C terminus
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