Tài liệu Báo cáo khoa học: The sensor protein KdpD inserts into the Escherichia coli membrane

The sensor protein KdpD inserts into the Escherichia coli membrane

independent of the Sec translocase and YidC

Sandra J. Facey and Andreas Kuhn

Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany

KdpD is a sensor kinase protein in the inner membrane of

Escherichia coli containing four transmembrane regions.

The periplasmic loops connecting the transmembrane

regions are intriguingly short and protease mapping allowed

us to only follow the translocation of the second periplasmic

loop. The results show that neither the Sec translocase nor

the YidC protein are required for membrane insertion of the

second loop of KdpD. To study the translocation of the first

periplasmic loop a short HAepitope tag was genetically

introduced into this region. The results show that also the

first loop was translocated independently of YidC and the

Sec translocase. We conclude that KdpD resembles a new

class of membrane proteins that insert into the membrane

without enzymatic assistance by the known translocases.

When the second periplasmic loop was extended by an

epitope tag to 27 amino acid residues, the membrane inser￾tion of this loop of KdpD depended on SecE and YidC. To

test whether the two periplasmic regions are translocated

independently of each other, the KdpD protein was split

between helix 2 and 3 into two approximately equal-sized

fragments. Both constructed fragments, which contained

KdpD-N (residues 1–448 of KdpD) and the KdpD-C

(residues 444–894 of KdpD), readily inserted into the

membrane. Similar to the epitope-tagged KdpD protein,

only KdpD-C depended on the presence of the Sec translo￾case and YidC. This confirms that the four transmembrane

helices of KdpD are inserted pairwise, each translocation

event involving two transmembrane helices and a periplas￾mic loop.

Keywords: Escherichia coli; membrane protein; protein

translocation; epitope tag.

The inner membrane protein KdpD of Escherichia coli is

involved in osmoregulation. It comprises of 894 amino acid

residues organized as two hydrophilic domains that are

separated by four closely spaced transmembrane regions [1].

KdpD is functionally related to other sensor kinases like

PhoR and EnvZ and shows a moderate sequence homology

in parts of the C-terminal domain with other sensor kinases.

In the membrane, the KdpD protein forms a homodimer,

which has been proposed to be required for the kinase

function [2]. The transmembrane regions are necessary for

signal perception because mutants in the transmembrane

regions have been found that are defective in the osmotic

response [3]. To understand how the transmembrane helices

or the periplasmic loops sense an osmotic signal a precise

knowledge of the topology and membrane insertion of these

hydrophobic regions is crucial. Intriguingly, the two peri￾plasmic loops separating the transmembrane regions com￾prise of only four and 10 amino acid residues, respectively.

Multi spanning membrane proteins contain several

hydrophobic regions linked by hydrophilic loops of various

lengths ranging from a few amino acids to several hundred

residues, e.g. in SecD [4]. Long periplasmic loops are

translocated by the ATP-driven Sec translocase, whereas

small loops may be translocated by a synergistic mechanism

without the Sec translocase as has been observed for the

double-spanning M13 procoat protein [5,6]. Based on

results from a functional approach [7], a Sec-independent

insertion has also been suggested for melibiose permease,

which has six short periplasmic loops. Gafvelin and von

Heijne [8] have shown, through studying a tandem

construction of leader peptidase that spans the membrane

four times, that short periplasmic loops of about 25 residues

were translocated independently of SecA, whereas long

loops of 250 residues required the SecA-driven translocase.

However, De Gier et al. [9] found by using the tightly

controlled SecE mutant strain, that the SecYE translocase

may be involved in the translocation of a 25 residue

periplasmic loop. The authors suggested that the hydro￾phobicity of the transmembrane region determines the

requirement of the Sec translocase.

Proteins that are destined to be translocated across or

inserted into the bacterial inner membrane are targeted to

the translocation sites by multiple mechanisms. In E. coli,

secretory proteins are targeted to the inner membrane by

means of the chaperone SecB, which directs the newly

synthesized protein to the SecAsubunit of the translocase

complex of the Sec pathway, and whose membrane￾integrated components are SecY, E, and G [10]. In contrast,

polytopic membrane proteins are targeted to the membrane

by an essential ribonucleoprotein complex that is closely

related to the eukaryotic signal recognition particle (SRP).

E. coli contains Ffh (P48), which together with 4.5S RNA,

Correspondence to A. Kuhn, Institute of Microbiology and

Molecular Biology, University of Hohenheim, 70599 Stuttgart

Germany. Fax: + 49 711 4592238, Tel.: + 49 711 4592222,

E-mail: andikuhn@uni-hohenheim.de

Abbreviations: HA, haemagglutinin; SRP, signal recognition particle;

IPTG, isopropyl 1-thio-b-D-galactoside; CCCP, carbonyl cyanide

p-chlorophenylhydrazone; pmf, proton motive force.

(Received 11 December 2002, accepted 20 February 2003)

Eur. J. Biochem. 270, 1724–1734 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03531.x