Mutant analysis of the XpsL protein, a component of the Type II Secretion Apparatus of Xanthomonas campestris pv.campestris

• Main Authors: Hsing-Ting Wang, 王幸婷
• Other Authors: Nien-Tai Hu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/28400029943200558681

碩士 === 國立中興大學 === 生物化學研究所 === 91 === In the type II secretion pathway of Xanthomonas campestris pv. campestris, protein products of the xps gene cluster are responsible for translocating extracellular proteins from periplasm to the milieu. The XpsL protein of 373 amino acid residues is a member of the type II secretion apparatus. It is anchored in the cytoplasmic membrane through a hydrophobic sequence located between the 215th-233rd residues with its N-terminus facing cytoplasm. The XpsL protein forms a ternary complex with two other cytoplasmic membrane proteins XpsM and XpsN. Association between the XpsL and XpsM is stronger than either one with the XpsN protein. Through its N-terminal domain, the XpsL protein also interacts with the cytoplasmic protein XpsE. In order to analyze its non-membrane-anchoring regions, I constructed random insertional mutant XpsL. Among four mutants obtained, only one with the insertion located between the 274th-275th residues caused significant secretion defect in the complementation assay for α-amylase secretion. It also interferes normal secretion. Western blot analysis indicates that the mutant XpsL protein content in the cell, when expressed alone, rose significantly relative to that of the wild type XpsL. Immunoprecipition and pull-down experiments reveal that the mutant XpsL protein exhibits stronger association with XpsE than the wild type XpsL. By performing XpsL-XpsM complex dissociation assay, I found that the mutant XpsL has slightly higher tendency than the wild type XpsL to dissociate from the XpsM protein. XpsL self-association shown by Strep-Tactin affinity chromatography for the functional XpsL protein was not affected by the mutation. To analyze the membrane-anchoring sequence of the XpsL, I mutated each of the hydrophobic amino acid residues to Cysteine. Each remains functional. Treatment with oxidizing agent followed by SDS-PAGE without β-mercaptoethanol (β-ME) and immunoblotting analysis reveal that the mutations located between the 215th-218th, 225th-227th and 230th-233rd residues caused the appearance of a β-ME-sensitive protein band of large size. It was speculated to be the disulfide-bonded XpsL dimer. The results imply that in the cell each XpsL is probably next to a different XpsL molecule in various directions.