Affinity-chromatography and yeast-two hybrid analysis of the complex formation among XpsE, -F, -L, -M and -N in xanthomonas campestris

• Main Author: 李振東
• Other Authors: 呂維茗
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/37870313336449191830

碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Abstact Xanthomonas campestris is an important phytopathogen in which the integrity of secretion machinery is critical for its pathogenicity. The type II secretion pathway (also known as the general secretion pathway) that requires the Sec proteins is known to exist in Xanthomonas. The type II secretion pathway is constituted by a stepwise process: secreted proteins are firstly translocated by sec-dependent mechanism through the inner membrane, released into the periplasm, and transported across the outer membrane by the so-called Gsp proteins. The Gsp proteins in Xanthomonas campestris contain at least 11 members. XpsE, with the ATP-binding motif, is a cytoplasmic protein associated with the cytoplasmic membrane through interaction with XpsL. XpsL, -M and -N are cytoplasmic membrane proteins that assembly as ternary complex. The stabilities of these three proteins are mutually dependent. XpsD is an outer membrane which forms the secretion pore. XpsG probably assemble with XpsH, -I, -J and -K and form pilus-like structure in periplasm. In contract to the above, roles of XpsF, a cytoplasmic membrane protein with two main domains facing inward, remain unknown thus far. It was speculated that XpsF may act, as platform for apparatus assemble or alternatively, as chaperon for protein folding. According to the predicated membrane topology, it is proposed that XpsF may interact with XpsE or XpsL, the other two Xps members with major cytoplasmic domains. Demonstrated by co-immunoprecipitation, XpsF was found to form complex with XpsE and XpsL/M/N, although only to a scarce amount under overexpressed condition. Using metal-affinity chromatography, we aimed to detect the complex formation among the above five proteins in strains with secretion ability. Furthermore, xps chromosomal mutants were examined to evaluate the possibility that the secretion complex may be locked and stabilized while the machinery is partially defective. However, only XpsL/M/N and XpsL/E complex were sucessfully detected by the above two approaches. Using the yeast two-hybrid system, we further dissect the interaction domains by removing the membrane-anchor region of XpsF, XpsL and XpsM.