Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni.
The small heat shock protein (smHSP) family has been extensively studied in eukaryotic cells. SmHSP assemble into large multimeric structures and possess chaperone activity that can prevent protein aggregation in vitro. Few studies on prokaryotic smHSP are actually available and no smHSP from lactic acid bacteria has been characterized at a biochemical level to date. Here we report on the Lo18 membrane-associated smHSP from the lactic acid bacterium Oenococcus oeni. Using size exclusion chromatography, nondenaturing pore-exclusion PAGE and in vitro and in vivo cross-linking experiments, the multimeric structure of Lol8 from O. oeni or expressed in Escherichia coli was investigated. In vitro, Lo18 is able to form a trimer and a higher oligomer which could be a dodecamer. Experiments strongly suggest that the same structures exist in vivo. First, Lo18 prevented thermal aggregation of citrate synthase and lactate dehydrogenase even at 60degreesC. These findings showed that the prokaryotic smHSP Lo18 can function as a molecular chaperone in vitro. Second, Lo18 did not protect lactate dehydrogenase from thermal inactivation and did not assist in enzymatic activity restoration after thermal aggregation, suggesting that other components may be needed for the refolding of the enzyme in an active conformation. Third, we showed that membrane association of Lo18 depends on the temperature upshift. Moreover, expression of this smHSP was induced by administration of a membrane fluidiser, the benzyl alcohol, suggesting that Lo18 expression could be regulated by the level of membrane fluidity.
Delmas
F
F
Laboratoire de Microbiologie UMR. INRA-Université, ENSBANA, Dijon, France.
Pierre
F
F
Coucheney
F
F
Divies
C
C
Guzzo
J
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006095:Gram-Positive Cocci / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007934:Leuconostoc / Q000235:genetics / Q000378:metabolism; D008560:Membrane Fluidity; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D002950:Citrate (si)-Synthase