Involvement of residues 96-104 of XpsN in its self-interaction examined by cysteine-scanning mutants analysis

• Main Authors: Cheng-Yi Tsao, 曹琤宜
• Other Authors: Nien-Tai Hu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/56505719134920014304

碩士 === 國立中興大學 === 生命科學系 === 93 === Abstract The XpsN pretien is one of the inner membrane proteins of type II secretion apparatus in Xanthomonas campestris pv. campestris. Near its N-terminus, there is a hydrophobic sequence spanning the cytoplasmic membrane once with a C-terminal periplasmic domain. Previous studies indicated that XpsN could associate with itself. In addition, it also interacts with other members of type II secretion apparatus, including the inner membrane protein complex XpsL-M, the outer membrane protein XpsD and the major pseudopilin XpsG. Thus, it was proposed to play a central role in type II secretion apparatus. Results from co-immune precipitation and GST pull down assay suggested that the sequence between the 97th to 110th residues of XpsN is required for its interaction with itself and with XpsD. To confirm significance of this region, I performed functional analysis of XpsN mutants with single amino acid at 96th-104th residues mutated to cysteine. By introducing each into the xpsN mutant strain of X. campestris XC1707 and assaying for their secretion, I found that T96C and V97C mutant proteins kept 60-90% secretion ability. While the secretion exhibited by TR98C, L99C and T100C mutants were redued to lower than 30%, G101C and L104C have entirely lost the ability. I further constructed four alanine substitution mutants at 99th, 100 th, 101st and 104 th residues and analyzed their function. They all possess higher secretion ability than cysteine substitution. The cysteine scanning mutants were furrher analyzed for dimerization by seperation them on SDS-polyacrylamide gel in absence of -mercaptoethanol. T96C, R98C, T100C, L104C mutants appeared to form disulfide-bonded dimmer with or without addition of iodoacetic acid before cell breakage. These obseravations suggest close proximity of the 96th, 98th, 100st and 104st residue of two neighboring XpsN in the secretion apparatus.