Antibodies against F1-ATPase alpha-subunit recognize mitochondrial chaperones. Evidence for an evolutionary relationship between chaperonin and ATPase protein families.

A Alconada,A.I Flores,L Blanco,J.M Cuezva

doi:10.1016/s0021-9258(17)36882-5

Abstract

Antibodies raised against two synthetic peptides from rat liver F1-ATPase alpha-subunit sequence recognized two main heat-shock proteins from Drosophila (p71 and p56) and rat liver (p74 and p54) cells. One of the antisera showed a 20-fold higher reactivity toward Escherichia coli GroEL chaperonin than toward the alpha-subunit purified from Drosophila. Indirect immunofluorescence microscopy and subcellular fractionation experiments located both mammalian heat-shock proteins in the mitochondria. The recent findings of functional homology between chaperonins and alpha-subunits, together with the unsuspected immunological reactivity of two mitochondrial molecular chaperones toward antisera derived from two different sequence motifs of the alpha-subunit, strongly argue in favor of the existence of an evolutionary relationship between chaperonins and alpha-subunits. The complete sequence alignment of F-type ATPase alpha-subunits and chaperonins revealed the existence of eleven most conserved regions (approximately 30% of each protein sequence) with an overall amino acid identity of 20 +/- 2% and similarity of 39 +/- 4%. A search of protein data bases with three different consensus sequences derived from this alignment identified a significant proportion of proteins belonging only to these two protein families. Since the alpha-subunit protein family is evolutionary related to the other catalytic (A and beta) and regulatory (B) subunits of V- and F-type ATPases, the homology reported herein allowed us to analyze, in the chaperonin sequences, the conservation of critical residues involved in nucleotide binding. These data support the hypothesis that chaperonins and the major subunits of V- and F-type ATPases are evolutionary related.

Highlights

From the Departamento de Biologta Molecular, Centro de Biologta Molecular “SeveroOchoa,” Consejo Superior de Investigaciones Cientificas-Universidad Aut6noma de Madrid (C.S.I.C.-U.A.M.), Universidad Aut6noma de Madrid, 28049 Madrid, Spain
Croscopy and subcellular fractionation experiments lo- assembly of other proteins being their best characterized role cated both mammalian heat-shock proteins inthe
The unsuspected immunological reactivity of two mito- The mitochondrial FIFO-ATPsynthase is a multisubunit prochondrial molecular chaperones toward antisera de- tein complexof the inner mitochondrialmembrane that couples rived from two different sequence motifs of the a-sub- the H+electrochemical gradient generated from the respiratory unit, strongly argue in favor of the existence of an chain to the synthesis of ATP (for reviews, see Amzel and Peevolutionary relationship between chaperonins and dersen (1983) and Pedersen and Carafoli (1987a and 1987b)

Summary

11 To whom correspondence should be addressed

Centrode Biologia mitochondrial protein import and processing. Provided further evidence that thea-subunit isinvolved in the Structural Identities between Chaperonins and a-F,-ATPases import of mitochondrial precursorasn, other functional activity glucose, 2 m~ glutamine, and 5% (w/v)of a mixture of essential amino in which molecular chaperones play a fundamental role. The existence of structural (Luis et al, 1990) and functional (Ami et al, 1991; Yuan and Douglas, 1992) similarities between chaperonin and a-subunit protein families raisesthe acids, a t 37 or 44 "C (heat-shocktreated), with continuous shaking (100 strokedmin) in 25-ml Erlenmeyer flasks with a mixture of 95%0,and 5% CO, as gas phase. The results ofimmunological experiments andcomplete sequence alignment stronglysupport the evolutionary relatedness of chaperonins and F,-ATPase a-subunits. The extension of these described (Cuezvaet al., 1990). Western Blot Determination of Immunoreactive Proteins-Aliquots of cellular (SL2cells), or mitochondrial, cytosolic and microsomal

EXPERIMENTAL PROCEDURES

RESULTS

VI VI1

Findings

DISCUSSION