Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 17 August 2007:
Vol. 317. no. 5840, pp. 961 - 964
DOI: 10.1126/science.1143993
Prev | Table of Contents | Next

Reports

Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine

Seokhee Kim1, Juliana C. Malinverni2, Piotr Sliz3,4, Thomas J. Silhavy2, Stephen C. Harrison3,4 and Daniel Kahne1,3*

1 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
2 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
3 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
4 Howard Hughes Medical Institute and Children's Hospital Laboratory of Molecular Medicine, Boston, MA 02115, USA.


Figure 1 Fig. 1. Diagram of bacterial outer membrane protein (OMP) biogenesis. [View Larger Version of this Image (45K GIF file)]
 

Figure 2 Fig. 2. Structure of YaeT. (A) Domain organization. (B) X-ray structure of YaeT21-351. POTRA domains P1 to P4 are colored yellow, green, blue, and red, respectively. The eight residues from P5 are colored gray. The missing electron density in the P3 domain is represented by a dashed line. (C) Ribbon diagram of a POTRA domain (P2) with side chains of the conserved residues shown. (D) Sequence alignments of POTRA domains from selected members of the YaeT/Omp85, Sam50, and Toc75 families, found in Gram-negative bacteria, mitochondria, and chloroplasts or cyanobacteria, respectively [adapted from Sánchez-Pulido et al. (14)]. Conserved residues are highlighted (28). The intensity of the orange color reflects the level of conservation in physicochemical properties. (E) X-ray structure of the dimer. The POTRA domains in one monomer are colored as in (B); the other monomer is purple. (F) Dimer interface showing the C-terminal residue contacts of one monomer (gray) to the P2 (light green) and P3 (light blue) domains of the other monomer. Labels represent hydrophobic residues. L, Leu; Y, Tyr; F, Phe; V, Val; I, Ile; T, Thr. [View Larger Version of this Image (63K GIF file)]
 

Figure 3 Fig. 3. (A) SDS-PAGE analysis of YaeT wild-type (wt) and deletion mutants from whole-cell lysates, without (–) and with (+) prior heat treatment. Proteins were detected by Western blot analysis with the use of an antibody recognizing the His tag. (B) His-tagged YaeT wild-type or deletion mutants ({Delta}P1 to {Delta}P5) and associated proteins following Ni-affinity chromatography. Eluted samples were blotted against His-tag, YfgL, NlpB, SmpA, and YfiO antibodies. (C) The purified YaeT complex run on a Blue-Native PAGE with molecular weights from a standard lane indicated. (D) Same as in (B), but YaeT was blotted with an antibody to YaeT. YaeT{Delta}P1 cannot be detected with our YaeT peptide antibody. (E) His-tagged wild-type YaeT and P3 mutants after purification by Ni-affinity chromatography and analysis, as in (B). [View Larger Version of this Image (61K GIF file)]
 

Figure 4 Fig. 4. Essentiality of POTRA domains. Cultures were grown with L-arabinose (A) or D-fucose (B) to induce or inhibit wild-type yaeT expression, which is driven by the ara PBAD promoter (5). Plasmid-borne yaeT variants were constitutively expressed. Samples taken after 6 hours were subjected to Western analysis. (A) Strains expressing plasmid-borne yaeT variants grew normally when wild-type yaeT was expressed. YaeT{Delta}P1 cannot be recognized with our YaeT peptide antibody (Fig. 3). Strains have low levels of DegP and normal OMP levels (LamB and OmpA). (B) When wild-type YaeT is absent, strains producing mutant YaeT variants exhibit growth defects. Strains expressing {Delta}P1 and {Delta}P2 grow better and have higher levels of OMPs than {Delta}P3, {Delta}P4, and the vector-only control. Although levels of {Delta}P1 cannot be quantified, {Delta}P2 is stable, indicating insertion into the membrane even in the absence of wild-type YaeT. Nevertheless, all strains lacking wild-type YaeT exhibit a strong extracytoplasmic stress response (increased DegP) indicative of OMP-assembly defects. Asterisk in (B) corresponds to proteolyzed DegP. OD, optical density. [View Larger Version of this Image (67K GIF file)]
 

To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)