Functional and molecular characterisation of two stomatin-like proteins from Arabidopsis thaliana

QH301 Biology;QK Botany;Q Science (General)

Gehl, Bernadette ; Blatt, Michael Robert

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: Band-7 protein family, stomatin, Arabidopsis thaliana, mitochondria, gene expression, subcellular localisation, transgenic plant, phenotype analysis;   

Others  :  http://theses.gla.ac.uk/640/1/2009Gehlphd.pdf

来源: University of Glasgow

【 摘 要 】

Stomatins belong to the band-7 (or SPFH domain) family (short for Stomatin, Prohibitin, Flotillin HflC/K) of diverse membrane proteins. This protein family is evolutionary conserved with members found in all sequenced eukaryotes and in most prokaryotes. Band-7 family proteins have the ability to oligomerise and generally aid in the assembly and regulation of large membrane-bound protein complexes. In animals, stomatins have been demonstrated to regulate ion channels by direct protein interactions. Additionally, they localise to membrane microdomains where they actively contribute to their assembly by binding sterols, and they also associate with the actin cytoskeleton. The Arabidopsis genome encodes for two structurally similar stomatin-like proteins that are functionally completely unknown yet. They will be referred to as AtSlp1 (for Arabidopsis thaliana stomatin-like protein) and AtSlp2. The aim of this thesis was to provide a detailed characterisation of these two genes on a molecular and functional level. Both proteins are expressed ubiquitously throughout plant development, but they accumulate at particularly high levels in pollen and other metabolically active cells. Phylogenetic analysis reveals that AtSlps are homologous to stomatin-like proteins of type 2. Amongst these, the human stomatin-like protein 2 (HsSlp2) is localised to mitochondria where it participates in large membrane-bound protein complexes and is also involved in the proliferation of cancer cells. Evidence is provided here that demonstrates mitochondrial localisation of both Arabidopsis Slp proteins in vitro and in vivo. On a functional level, mitochondria from an slp1 knockout mutant plant have a decreased mitochondrial membrane potential and increased oxygen consumption rates. This is interpreted as a defect in coupling efficiency and an impairment of the mitochondrial inner membrane integrity. This defect results in a variety of other growth phenotypes that are related to metabolically active tissues and cell types. Knockout plants are delayed in overall growth of shoots and roots and have decreased seed germination rates. Additionally, these plants are less resistant to conditions of high salinity and are less fertile. Overexpression of a protein acting as a putative dominant-negative Slp fragment results in plants with a dwarf phenotype and early onset of leaf senescence. This phenotype correlates with increased levels of reactive oxygen species and altered organelle ultrastructure. Guard cells from these plants in particular have enlarged chloroplasts and are impaired in transpirational control. It is concluded that also in plants, stomatins act together with other band-7 family proteins as parts of large protein complexes that have regulatory roles important for development and stress responses. Their main role is probably to provide membrane scaffolds that affect mitochondrial function and morphology during cell division and in situations of mitochondrial stress.