Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Biol Chem. 2008 Aug;389(8):1107-15. doi: 10.1515/BC.2008.122.

In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells.

Author information

  • 1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 507 PCTB, 725 North Wolfe Street, Baltimore, MD 21205, USA. surban@jhmi.edu

Abstract

Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.

PMID:
18979634
[PubMed - indexed for MEDLINE]
PMCID:
PMC2678756
Free PMC Article

Images from this publication.See all images (8)Free text

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for iFactory Icon for PubMed Central
    Loading ...
    Write to the Help Desk