Tài liệu Báo cáo khóa học: Further insights into the assembly of the yeast cytochrome bc1 complex

Further insights into the assembly of the yeast cytochrome bc1

complex based on analysis of single and double deletion mutants

lacking supernumerary subunits and cytochrome b

Vincenzo Zara1

, Ilaria Palmisano1

, Laura Conte1 and Bernard L. Trumpower2

1

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita` di Lecce, Italy; 2

Department of Biochemistry,

Dartmouth Medical School, Hanover, NH, USA

The cytochrome bc1 complex of the yeast Saccharomyces

cerevisiae is composed of 10 different subunits that are

assembled as a symmetrical dimer in the inner mitochondrial

membrane. Three of the subunits contain redox centers and

participate in catalysis, whereas little is known about the

function of the seven supernumerary subunits. To gain fur￾ther insight into the function of the supernumerary subunits

in the assembly process, we have examined the subunit

composition of mitochondrial membranes isolated from

yeast mutants in which the genes for supernumerary sub￾units and cytochrome b were deleted and from yeast

mutants containing double deletions of supernumerary

subunits. Deletion of any one of the genes encoding cyto￾chrome b, subunit 7 or subunit 8 caused the loss of the other

two subunits. This is consistent with the crystal structure

of the cytochrome bc1 complex that shows that these three

subunits comprise its core, around which the remaining

subunits are assembled. Absence of the cytochrome b/sub￾unit 7/subunit 8 core led to the loss of subunit 6, whereas

cytochrome c1, iron–sulfur protein, core protein 1, core

protein 2 and subunit 9 were still assembled in the mem￾brane, although in reduced amounts. Parallel changes in the

amounts of core protein 1 and core protein 2 in the mito￾chondrial membranes of all of the deletion mutants suggest

that these can be assembled as a subcomplex in the mito￾chondrial membrane, independent of the presence of any

other subunits. Likewise, evidence of interactions between

subunit 6, subunit 9 and cytochrome c1 suggests that a

subcomplex between these two supernumerary subunits and

the cytochrome might exist.

Keywords: cytochrome bc1; assembly; supernumerary sub￾units; Saccharomyces cerevisiae.

The cytochrome bc1 complex is a multisubunit complex

embedded in the inner membrane of mitochondria [1,2].

This respiratory enzyme catalyzes the transfer of electrons

from ubiquinol to cytochrome c and couples the electron

transfer to vectorial proton translocation across the inner

mitochondrial membrane. The bc1 complex has been

crystallized and analyzed from bovine, chicken and yeast

mitochondria [3–7].

In mitochondria of the yeast Saccharomyces cerevisiae,

the cytochrome bc1 complex is composed of 10 different

subunits organized in the lipid bilayer as a homo-dimer as

shown in Fig. 1A [8,9]. There are three catalytic subunits

that contain redox prosthetic groups, cytochrome b, cyto￾chrome c1 and the Rieske iron–sulfur protein (ISP). In

addition, there are seven supernumerary subunits that lack

any cofactors.The supernumerary subunits are core protein 1

and core protein 2 [10,11], with apparent molecularmasses of

44 and 40 kDa on SDS/PAGE, respectively, and five

smaller proteins. The latter are Qcr6p [12], Qcr7p [13], Qcr8p

[14], Qcr9p [15] and Qcr10p [8] with apparent molecular

masses of about 17, 14, 11, 7.3 and 8.5 kDa, respectively.

Although the supernumerary subunits of the mitochond￾rial bc1 complexes were discovered one to two decades ago

[16], little is known about their function. It is also not known

how these peripheral subunits are assembled around the

catalytic core of the enzyme to arrive at the three dimen￾sional organization revealed by the crystal structures

(Fig. 1A). The supernumerary subunits and the catalytic

subunits of the yeast cytochrome bc1 complex show

sequence similarities to those of the bc1 complexes of higher

eucaryotes [1,2,9]. In addition, the crystallographic analysis

of the Saccharomyces cerevisiae cytochrome bc1 complex

has revealed an essentially identical overall structure of this

complex and that of chicken and beef [6]. In yeast and

higher eukaryotes, cytochrome b is encoded by mito￾chondrial DNA, while the remaining subunits of the bc1

complex are encoded in the nucleus, synthesized by cytosolic

polysomes, and then imported into mitochondria, thereby

reaching their final location in the inner membrane [17]. The

similarities of the yeast bc1 complex to the bc1 complexes

of higher eukaryotes suggest that the yeast enzyme may

serve as a paradigm to understand how this oligomeric

protein complex is assembled into the inner mitochondrial

membrane.

Correspondence to V. Zara, Dipartimento di Scienze e Tecnologie

Biologiche ed Ambientali, Universita` di Lecce, Via Prov.le Lecce￾Monteroni, I-73100 Lecce, Italy. Fax: + 39 0832 298626,

Tel.: + 39 0832 298705, E-mail: [email protected]

Abbreviations: DFP, diisopropyl fluorophosphate; ISP, Rieske

iron–sulfur protein.

(Received 8 January 2004, revised 23 January 2004,

accepted 6 February 2004)

Eur. J. Biochem. 271, 1209–1218 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04024.x