Gas vesicle formation of haloarchaea: functions of the accessory Gvp proteins.

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Gas vesicles are intracellular nanostructures consisting of a proteinaceous wall surrounding a gas-filled space. GvpA and GvpC are the major constituents of this wall. GvpA forms antiparallel dimers that aggregate into ribs running as low-pitch helix perpendicular to the long axis. GvpC stabilizes the gas vesicle structure by attaching to the exterior surface. Ten additional Gvp proteins encoded by the early transcription unit gvpFGHIJKLM and the gvpACNO gene cluster are produced in minor amounts, and most of them are essential for gas-vesicle assembly. These accessory Gvp proteins might form protein-protein complexes required at the onset or during gas-vesicle formation. To investigate protein-protein interactions in haloarchaea we successfully adapted the split-GFP method. In addition, protein complexes were selected from haloarchaea using the cellulose-binding domain, CBD. The GvpM-GvpL interaction was confined to the N-terminal 25-aa of GvpM by split-GFP. Also, GvpH and GvpJ bind GvpM, and we propose to determine the binding sites of both in GvpM. If the binding sites of the three accessory Gvp overlap in GvpM, it is likely that they bind consecutively. A GvpM-CBD fusion selected GvpF, GvpH, GvpJ, GvpK, and GvpL from Hfx. volcanii transformants. These Gvp proteins appear to form a complex that might serve as nucleation site for the directed aggregation of GvpA at the onset of gas-vesicle assembly. We propose to study the FHJKLM-complex(es) to determine their size(s) and constituents. GvpF and GvpK are both constituents of this complex, and we propose to investigate whether they bind GvpM directly or whether other Gvp proteins mediate their interaction. Also, we will test the formation (and enlargement) of this complex in the presence of minor amounts of GvpA. GvpG and GvpI were not found so far. CBD-tagged GvpG and GvpI will be used to select putative binding partners. In addition, the interaction network of GvpA will be studied using fragments of this protein. Helix 1 will be tested as putative binding site between two ribs, and helix 2 might serves as regular anchor site of GvpC at the exterior surface of the gas vesicle wall. Also, the putative function of GvpN as AAA-ATPase will be investigated by substituting amino acids of the p-loop motif found in this protein.