Caveolin Revealed: A Mutagenesis Study of Caveolin-1

Abstract

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), an 81 kDa GTPase, is the main mediator of mitochondrial fission. In order to mediate fission, Drp1 is thought to form large oligomers in the presence of nucleotide on the outer mitochondrial membrane. Using sedimentation assays and electron microscopy, we confirm that Drp1 selfassembles in the presence of either or both nucleotide and artificial lipid bilayers in vitro. We further identify these oligomeric species as conformationally distinct structures. Drp1 constricts lipid bilayers through a mechanism that requires GTP hydrolysis. Following constriction, Drp1 disassembles and can reassociate with lipid bilayers. Here we also show that the variable domain (VD) is not required for lipid association. Rather, it limits Drp1 oligomerization and aids in oligomer curvature. Our results support the conclusion that Drp1 is a mechanoenzymatic protein regulated through distinct interactions with nucleotide and liposomes. 1865-Pos Board B2Caveolin Revealed: A Mutagenesis Study of Caveolin-1 Sarah Plucinsky, Kerney J. Glover. Lehigh University, Bethlehem, PA, USA. Caveolae are 50-100 nm invaginations in the plasma membrane that are rich in cholesterol, sphingomyelin and the integral membrane protein called caveolin-1. Caveolin-1 has three main functions: forming caveolae, cell signaling, and endocytosis. To examine the importance of conserved residues within the scaffolding and intra-membrane domains of caveolin-1, alanine and phenylalanine scanning mutagenesis was performed on conserved residues. These residues were identified by sequence alignment of the three caveolin isoforms. Sixteen residues (S88, F92, K96, Y97, Y100, L103, P110, A112, G116, F119, A120, S123, H126, I127, W128 and P132) were mutated individually to both alanine and phenylalanine and subjected to analysis by NMR spectroscopy (1H-15N-HSQC). The effect of the mutation on the overall protein structure was monitored by comparison to the wild-type spectrum. These mutagenesis studies reveal seven residues (Y100, P110, A112, G116, S123, H126, and P132) that are structurally significant. The residues within the scaffolding domain are more permissive to mutation than the intra-membrane domain. The proline residues within the intra-membrane domain were shown to be critical for protein structure. These findings shed light onto the structurally relevant residues within the caveolin-1 scaffolding and intra-membrane domains, and further our understanding of the requirements for protein structure and stability.