Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli.
Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.
Sakoh
Machiko
M
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Ito
Koreaki
K
Akiyama
Yoshinori
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism*; D045726:Metalloproteases; D011233:Precipitin Tests; D011489:Protein Denaturation; D020673:Protein Renaturation / Q000187:drug effects; D000069816:SEC Translocation Channels; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D002364:Caseins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C068607:HtpX protein, E coli; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D045726:Metalloproteases; D015032:Zinc