hcred, a genetically encoded fluorescent binary cross-linking agent for cross-linking of mitochondrial atp synthase in saccharomyces cerevisiaehcr、基因编码的荧光二进制交联剂交联的线粒体atp合酶在酿酒酵母.pdfVIP

HcRed, a Genetically Encoded Fluorescent Binary Cross- Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae 1,2 1 1,2 1 Lan Gong , Georg Ramm , Rodney J. Devenish , Mark Prescott * 1 Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Victoria, Australia, 2 ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton Campus, Victoria, Australia Abstract Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross- linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial