Molecular Characterisation of Ductin, the Membrane Component of the Gap Junction and the Vacuolar Proton Pump

[摘要] This thesis describes an investigation into ductin, a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H+- ATPase (V-ATPase) and a unique component of the connexon channel of gap junctions. The aim of the thesis was to identify regions of ductin involved in the association with other subunits of the V-ATPase and to determine the manner of ductin's synthesis and assembly into a multimeric state. Ductin has been studied using a cell-free expression system based on rabbit reticulocyte lysate and in a yeast system using a strain of conditional lethal yeast that are deficient in ductin. A set of residues thought to be critical for the activity of ductin reside in the two conserved extramembranous loop regions, which are thought to serve as sites of contact with other subunits of the V-ATPase complex. To test their importance these loop regions were exchanged with the corresponding regions of VMA11, protein homologous to ductin but with a distinct activity. The resulting loop chimeras of ductin were expressed in the yeast strain deficient in ductin and the activity of the chimeras was assayed. Both of the conserved loop regions were found to be sensitive to substitution, with the first loop between helices 1 and 2 having a significantly reduced activity while the second loop between helices 3 and 4 was inactive. This indicates that these conserved loop regions are important for the activity of ductin, possibly in a manner similar to that of the polar loop region of the F1F0-ATPase subunit c. The synthesis of ductin was dependant on the presence of microsomal membranes, and ductin was inserted into this endoplasmic reticulum derived membrane, as shown by trypsin insensitivity and sodium carbonate extraction. The self-assembly of ductin into an oligomeric complex was shown by the co-immunoprecipitation of two forms of ductin using antibodies specific to one of the ductin forms. The presence of an aqueous channel in this oligomeric complex was shown by the labelling of cysteine replacement mutants in the putative central gap junctional chanel with a membrane impermiant maleimide probe, fluoroscein-5-maleimide. The orientation of ductin once it is inserted into the microsomal membranes was determined by two methods, firstly from the sensitivity to trypsin of a C-terminal extension, and secondly from the labelling by fluoroscein-5-maleimide of single cysteine residues. A trypsin sensitive C-terminal extension based on the mature N-terminus of beta-lactamase was engineered onto ductin (Nductin-beta lac) with the expectation that the ability of trypsin to cleave this extension would be determined by the orientation of ductin. After trypsin treatment two populations of Nductin-beta lac were observed, one cleaved the other uncleaved, corresponding to a cytoplasmic and lumenal C-terminal location respectively. The presence of dual orientations for ductin was confirmed by the fluoroscein-5-maleimide labelling, since a single cysteine residue on either side of the microsomal membrane was only partially labelled while the membranes remained impermiant to the fluoroscein-5-maleimide.

[发布机构] University:University of Glasgow

[关键词] Molecular biology, Cellular biology