Tài liệu Báo cáo khoa học: The role of the ESSS protein in the assembly of a functional and stable

The role of the ESSS protein in the assembly of a functional and stable

mammalian mitochondrial complex I (NADH-ubiquinone

oxidoreductase)

Prasanth Potluri, Nagendra Yadava and Immo E. Scheffler

Division of Biology, Molecular Biology Section, University of California, San Diego, California, USA

The ESSS protein is a recently identified subunit of mam￾malian mitochondrial complex I. It is a relatively small

integral membrane protein (122 amino acids) found in the

b-subcomplex. Genomic sequence database searches reveal

its localization to the X-chromosome in humans and mouse.

The ESSS cDNA from Chinese hamster cells was cloned and

shown to complement one complementation group of our

previously described mutants with a proposed X-linkage.

Sequence analyses of the ESSS cDNA in these mutants

revealed chain termination mutations. In two of these

mutants the protein is truncated at the C-terminus of the

targeting sequence; the mutants are null mutants for the

ESSS subunit. There is no detectable complex I assembly

and activity in the absence of the ESSS subunit as revealed by

blue native polyacrylamide gel electrophoresis (BN/PAGE)

analysis and polarography. Complex I activity can be re￾stored with ESSS subunits tagged with either hemagglutinin

(HA) or hexahistidine (His6) epitopes at the C-terminus.

Although, the accumulation of ESSS-HA is not dependent

upon the presence of mtDNA-encoded subunits (ND1-

6,4 L), it is incorporated into complex I only in presence of

compatible complex I subunits from the same species.

Keywords: complex I; ESSS protein; mitochondria; NADH￾ubiquinone oxidoreductase; respiration-deficient mutants.

NADH-ubiquinone oxidoreductase (complex I) is the first

enzyme in the mitochondrial electron transport chain

responsible for the oxidation of NADH. The complex I

from bovine heart is composed of 46 distinct subunits, of

which 14 have been assigned to the core complex, as

homologous subunits are found in the prokaryotic complex

capable of carrying out the same known functions: NADH

oxidation and establishment of a membrane potential by

proton translocation [1–6]. The precise role of the other 32

subunits is largely unknown, although some of these

(MWFE, the acyl carrier protein) have been shown to be

absolutely essential for assembly and function of the

complex [7–14].

No crystal structure is available for complex I; its overall

boot-shaped conformation has been deduced from low￾resolution electron microscopic studies [15–18]. In the

bovine complex a large subdomain is made up of
20

integral membrane proteins contributing > 60 transmem￾brane segments. Some of these must be intimately involved

in proton pumping. Another large subdomain is attached to

the membrane-subcomplex via a narrower neck-shaped

domain. This peripheral-subcomplex contains a flavin

mononucleotide and at least seven iron sulfur centers

involved in electron transport from NADH to ubiquinone.

A major challenge is to understand how electron transport

is coupled to proton pumping.

Structure–function analyses of electron transport com￾plexes have in the past been advanced considerably by a

combination of biochemical and genetic studies, largely

carried out with the bovine complex I [1,2,19–22]. Complex

I lags behind, largely because a similar complex does not

exist in the common yeasts Saccharomyces cerevisiae and

Schizosacchoromyces pombe. Genetic studies with Neuros￾pora crassa [11], and more recently with the yeast Yarrowia

lipolytica [23] and the unicellular algae Chlamydomonas

[24,25] have provided some notable insights.

Finding mutations in mammalian systems affecting

complex I has been even more of a challenge. A systematic

investigation of human patients suffering from mitochond￾rial diseases has led to the characterization of human cell

lines with partial complex I deficiency. Such cell lines can be

subdivided into those with mutations in the mitochondrial

genome [26], and those with mutations in nuclear genes

[27–30].

Our laboratory has described a series of respiration

deficient Chinese hamster cell mutants with very severe or

complete defects in complex I activity [31–34]. A genetic

analysis by somatic cell hybridization has revealed the

existence of several complementation groups, and it has

been proposed that more than one of these genes are

X-linked [35]. These early conclusions were confirmed for

one complementation group in which a defect in the

Correspondence to I. E. Scheffler, Division of Biology, Molecular

Biology Section, University of California, San Diego, CA 92093–0322,

USA. Fax: + 1 858 5340053, Tel.: + 1 858 5342741,

E-mail: [email protected]

Abbreviations: BN/PAGE, blue native polyacrylamide gel electro￾phoresis; MBS, maleimidobenzoyl N-hydroxysuccinimide ester;

TMPD, tetramethylphenylene diamine.

(Received 10 March 2004, revised 10 June 2004,

accepted 18 June 2004)

Eur. J. Biochem. 271, 3265–3273 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04260.x