Abstract
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.
Highlights
In E. coli, only a few other membrane-integrated proteases are known: DegS and RseP (YaeL), which introduce regulated cleavage into a membrane-bound substrate [11,12,13], and Lep, which cleaves off a signal peptide of secretory precursor proteins [14]
Our results showed that ftsH disruption led to the induction of the Cpx stress response, which was exaggerated further by overexpression of SecY or Fo subunit a in the absence of FtsH [18]
We have described some biochemical properties of HtpX and confirmed its proteolytic activity
Summary
Bacterial Strains and Media—E. coli K12 strains AD16 (FЈlacIq) [19], AD202 (ompT::kan) [20], SC1060 (AD16, sfhC21, zad-220::Tn10) [18], and AD1691 (SC1060, ftsH3::kan) [18] have been described previously; MA90 (SC1060, htpX::tet) and MA91 (AD1691, htpX::tet) were constructed as follows. The htpX::tet marker was introduced into AD16 carrying pKN201 (Ptac-red-gam) Murphy) by linear transformation [21] using a DNA fragment amplified from the chromosomal DNA of AD1735 (zad-220::Tn10) with a pair of primers (5Ј-CGCATATTGCGTTTTGTTAAACTGAGGTAAAAAGAAAATTCCGACCTCATTAAGCAGCTC-3Ј and 5Ј-GCGCGTCGATCAGGACGCGCTTTTTAGTATTTACTTCATAAACTAAGCACTTGTCTCCTG-3Ј). The htpX::tet marker was P1-transduced into SC1060 and AD1691, respectively. L [22] and M9 [23] were used as complete nutrient and minimal salt media, respectively. Akiyama, unpublished results. 3 The abbreviations used are: IPTG, isopropyl-1-thio--D-galactopyranoside; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
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