| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 89 === Abstract
Gram-negative bacteria have developed different pathways for the secretion of extracellular proteins into melieu. In type II secretion pathway, proteins are secreted in two steps. They are first translocated across cytoplasmic membrane via the Sec machinery. In a second step it takes between 12-14 proteins for the proteins to traverse across the outer membrane. These proteins are designated XpsD-O in Xanthomonas campestris pv. campertris. The xpsM gene, which encodes a protein of 213 amino acid residues with two cysteines and two leucine-zipper like motif, is the subject of this study. The XpsM protein possesses an N-terminal hydrophobic region. It was predicted to be an integral inner membrane protein and was shown to form complex with XpsL and with XpsN. In this study, I performed gel filtration analysis of the XpsM-His protein in the absence (in XC17433) and in the presence (in XC1714) of other Xps proteins that were extracted with b-OG. The XpsM protein in XC17433 appeared as homomultimer of 10-12 subunits. However, it appeared as monomer in the XpsL-XpsM-XpsN complex when expressed from a plasmid- encoded gene in XC1714. SDS-PAGE analysis of XpsM in the absence of b-mercaptoethanol (b-ME) revealed a signal with twice the size of the monomer on the immunoblot, suggesting the involvement of cysteines in dimer formation. When I mutated both cysteines through site-directed mutagenesis, the b-ME sensitive XpsM dimer disappeared. However, it remains functional. This result suggested that disulfide bond formation is probably not essential for normal function of XpsM protein. I further analyzed the XpsM fusion protein L74::PhoA for its interactions with XpsL and with XpsN. Coimmuno- precipitation experiments revealed that XpsM remains capable of forming complex with XpsL and XpsN by keeping its N-terminal seventy-four amino acid residues.
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