No abstract

Telomeres are DNA repeats at the ends of linear chromosomes and are replicated by telomerase, a ribonucleoprotein reverse transcriptase. Telomere length regulation and chromosome end capping are essential for genome stability and are mediated primarily by the shelterin and CST complexes. POT1-TPP1, a subunit of shelterin, binds the telomeric overhang, suppresses ATR-dependent DNA damage response, and recruits telomerase to telomeres for DNA replication. POT1 localization to telomeres and chromosome end protection requires its interaction with TPP1. Therefore, the POT1-TPP1 complex is critical to telomere maintenance and full telomerase processivity. The aim of this mini-review is to summarize recent POT1-TPP1 structural studies and discuss how the complex contributes to telomere length regulation. In addition, we review how disruption of POT1-TPP1 function leads to human disease.

Caveolin-1 is an integral membrane protein that is the primary component of cell membrane invaginations called caveolae. While caveolin-1 is known to participate in a myriad of vital cellular processes, structural data on caveolin-1 of any kind is severely limited. In order to rectify this dearth, secondary structure analysis of a functional construct of caveolin-1, containing the intact C-terminal domain, was performed using NMR spectroscopy in lyso-myristoylphosphatidylglycerol micelles. Complete backbone assignments of caveolin-1 (residues 62-178) were made, and it was determined that residues 62-79 were dynamic; residues 89-107, 111-128, and 132-175 were helical; and residues 80-88, 108-110, and 129-131 represent unstructured breaks between the helices.

The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample. To demonstrate the utility of employing PFOA, we expressed and purified a segment of the extremely hydrophobic membrane protein caveolin-1.

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.