Hu Ping, Bian Zhuan, Fan Mingwen, Huang Meijing, Zhang Ping
The Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China.
Arch Oral Biol. 2008 Feb;53(2):150-4. doi: 10.1016/j.archoralbio.2007.08.008. Epub 2007 Oct 23.
The general secretory (Sec) pathway is the main pathway for protein transport. Sortase A has been shown to anchor surface protein containing an LPXTG motif to the cell wall. In order to know the distribution pattern of the Sec translocons and sortase A in Streptococcus mutans, we explored the subcellular localisation of SecA and sortase A.
The anti-SecA antibody and anti-sortase A antibody were examined on Western blots to evaluate specificities. An indirect post-embedding immunogold method was used to determine the subcellular localisation of the Sec pathway and sortase A enzyme in S. mutans membrane.
Immunoblotting experiments showed that the anti-SecA antibody and anti-sortase A antibody specifically recognised a single band of about 95 kDa and 27 kDa, respectively in the S. mutans GS-5 cell lysates. Immunogold electron micrographs showed that small and large gold particles clustered together at a distinct microdomain.
These data showed for the first time that Sec translocase and sortase A colocalised to a single locus in the cytoplasmic membrane of S. mutans GS-5.
一般分泌(Sec)途径是蛋白质运输的主要途径。分选酶A已被证明可将含有LPXTG基序的表面蛋白锚定到细胞壁上。为了了解变形链球菌中Sec转运体和分选酶A的分布模式,我们探究了SecA和分选酶A的亚细胞定位。
在蛋白质免疫印迹法中检测抗SecA抗体和抗分选酶A抗体,以评估其特异性。采用间接包埋后免疫金标法确定Sec途径和分选酶A在变形链球菌细胞膜中的亚细胞定位。
免疫印迹实验表明,抗SecA抗体和抗分选酶A抗体分别在变形链球菌GS-5细胞裂解物中特异性识别一条约95 kDa和27 kDa的单条带。免疫金电子显微镜照片显示,小的和大的金颗粒聚集在一个独特的微区。
这些数据首次表明,Sec转运体和分选酶A共定位在变形链球菌GS-5细胞质膜的单个位点上。