Proteolytic Activity of HtpX, a Membrane-bound and Stress-controlled Protease from Escherichia coli

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 selfdegradation 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 Zn 2؉ , 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.It is vital for cells that membranes and membrane proteins retain their integrity. Malfolded and misassembled membrane proteins, produced under stressful conditions and in non-physiological situations, should receive proteolytic quality control. In Escherichia coli, FtsH, a membrane-bound and ATP-dependent zinc metalloprotease, is known to play a central role in the degradation of unstable membrane proteins (1, 2). Thus, it degrades SecY, a subunit of protein translocase (3), and Fo subunit a of proton ATPase (4) when they have failed to assemble. It also degrades YccA, the function of which is unknown (5), and some unstable cytosolic proteins (1, 2). A characteristic feature of FtsH is that it processively degrades membrane-protein substrates by recognizing their ends when they protrude sufficiently into the cytosol (6, 7). This degradation appears to be accompanied by dislocation of the substrate from the membrane, a process presumably mediated by the ATPase function of .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 (11-13), and Lep, which cleaves off a signal peptide of secretory precursor proteins (14). HtpX was first described by Kornitzer et al. (15) as a heat-inducible protein from E. coli with sequence features of a membrane protein and a metalloprotease. They showed that its expression, in a form truncated at the C-terminal, enhanced degradation of cellular proteins in puromycin-treated cells (15). Subsequently, it was also shown that the htpX gene in Xylella fastidiosa was induced by an increase in temperature (16), whereas the Streptococcus gordonii htpX was not heat-inducible (17). Disruption of S. gordonii htpX caused changes in several properties of the cell surface, although the relationship of these changes with any protease activity of HtpX was unclear (17).We rediscovered HtpX during the course of study of cellular responses to "membrane protein stress." Our results showed that ftsH disruption led to the induction of the Cpx stress response, which wa...

Expression and purification of integral membrane metallopeptidase HtpX

“…The position of the characteristic HEXXH zinc-binding motif reveals that the catalytic moiety is most likely to be found on the cytosolic side of the membrane. This is in agreement with studies reporting that HtpX cleaves only the cytoplasmic regions of membrane protein SecY in vivo [16] and contrasts with Oma1, another family-M48 IMMP involved in maintaining the integrity of the mitochondrial inner membrane, which cleaves protein substrates on both sides of the membrane [17]. According to the zinc-binding motif, H 139 and H 143 most likely coordinate the catalytic zinc ion in HtpX.…”

“…Previously, wild-type HtpX had been reported to undergo rapid self-cleavage after homologous recombinant overexpression during cell disruption and/or membrane solubilization with detergent [16]. The protein was therefore purified under denaturing conditions and refolded in the presence of a metal chelator [16].…”

“…This vector confers resistance towards ampicillin and attaches a 4-kDa C-terminal tag comprising a factor Xa peptidase recognition site and a hexahistidine (His 6 )-tag among other residues [16].…”

“…After refolding, the protein proved to be an active zinc-dependent MP [16]. Here we have developed a bacterial overexpression system and a purification protocol …”

“…Another member of this latter family is HtpX, which was initially described as a heat-shock, Cpx-controlled protein [12,13]. HtpX is widespread in bacteria and archaea, and it participates in the proteolytic quality control of membrane proteins together with a two-pass transmembrane ATP-dependent MP, FtsH [14][15][16]. Disruption mutations of HtpX and FtsH revealed that they are essential for Escherichia coli growth and can compensate for each other when one of them is inactivated [13].…”