BIOCHEMICAL

Vol. 169, No. 2, 1990 June 15, 1990

MITOCHONDRIAL

IMPORT

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 391-396

OF THE HUMAN CHAPERONIN

(HSP60) PROTEIN

Bhag Singh, Hasmukh V. Patel, Robert G. Ridley, Karl B. Freeman and Radhey S. Gupta* Department of Biochemistry, McMaster University 1200 Main Street West, Hamilton, Ontario Canada L8N 325 Received

March

28,

1990

The mitochondrial import of a member of the “chaperonin” group of proteins which play an essential role in the import of protein into organelles and their subsequent proper folding has been examined. The cDNA for human hsp60 (synonyms: GroEL homolog, Pi) was transcribed and translated in vitro and its import into isolated rat heart mitochondria examined. The protein was converted into a mature form of lower molecular mass (= 58 kDa) which was resistant to trypsin treatment. The import of human hsp60 into mitochondria was inhibited in the presence of an uncoupler and also no import occurred when the N-terminal presequence was lacking. These results indicate that the chaperonin protein(s) are transported into mitochondria by a process similar to other imported mitochondrial proteins. Our results also indicate that although the Pl protein precursor was efficiently imported into mitochondria, in of other mitochondrial proteins (viz. ornithine comparison to precursors carbamoyltransferase and uncoupling protein) much less binding of pre Pl to mitochondria was observed. The significance of this latter observation at present is Q1990Academic Press, Inc. unclear.

The term “chaperonin” refers to a novel family of highly conserved, ubiquitous proteins which play an essential role in the intracellular folding and assembly of various proteins and protein complexes the GroEL carboxylase;

protein

(l-3).

of Escherichia

RBUp2)

ovary

(CHO)

co/i (1,4,5),

subunit binding protein

hsp60 protein of yeast mitochondria hamster

The members

of the chaperonin

rubisco

(RBUp2BP)

family include

(ribulose-l,5-bisphosphate of plant chloroplast

(1,2,6),

(7,8) and the Pl protein of human and Chinese

cell mitochondria

(9,lO).

Based

on extensive

sequence

homology, the 60-65 kDa major antigenic protein of various pathogenic bacteria (e.g. Mycobacterium leprae, M. tuberculosis, Coxiella burnetii, Ricketssia fsufsugamush~ is also a member of the chaperonin family (see 9-12). Several of the chaperonin group of proteins have been shown to carry out an essential “molecular chaperone” function

“To w.hom correspondence

should

be addressed. 0006-291X&O 391

$1.50

Copyright 0 1990 by Academic Press, inc. All rights of reproduction in any form reserved.

Vol.

169,

No.

2, 1990

(i.e. facilitating

BIOCHEMICAL

proper and preventing

AND

BIOPHYSICAL

improper

and assembly of proteins and protein complexes, include the requirement

RESEARCH

interactions)

COMMUNICATIONS

in the intracellular

respectively

(l-3).

folding

Examples of this

of the GroEL protein in phage head morphogenesis

and for

proper folding of functional rubisco in recombinant E. co/i cells and in vitro (see 1,35); involvement of RBUp2BP in the assembly of rubisco in plant chloroplasts (1,2,5,6); requirement

of hsp60 in yeast for import into mitochondria

assembly into oligomeric In eukaryotic

protein complexes

cells the members

such as mitochondria

(7,13).

of the chaperonin

and chloroplasts

of proteins and then their

family are located in organelles

which are presumed

to have evolved from

prokaryotes via endosymbiosis (14). This latter view is also supported by the sequence data on various eukaryotic and prokaryotic chaperonins (15). Our earlier studies on mammalian

Pl protein indicated that it is encoded by a nuclear gene, and imported

the mitochondrial

into

matrix (16). These earlier studies also showed that Pl is synthesized

as a precursor molecule whose maturation is inhibited by the potassium ionophores nonactin and valinomycin (16), which dissipate the mitochondrial membrane potential essential for protein import (13,17). Chinese hamster

Recently the cDNAs for hsp60 from human

(10) and yeast (18) cells have been cloned and sequenced.

contain a N-terminal leader or presequence with the potential of forming charged amphiphilic a-helix, considered essential for mitochondrial import

(9), They

a positively (13,171. In

the present communication, we have examined the import of in vitro synthesized human hsp60 (Pl) protein into isolated rat heart mitochondria and the necessity of the presequence MATERIAL

for this process. AND METHODS

Construction of cDNA clones. The clone xC5 (an isolate from a xgtll library) which contains the complete cDNA sequence of human Pl protein (9) was digested with the flanking enzymes Kpn I and Sst I and the resultant 4.2 kb fragment was subcloned in the plasmid pTZ-18U (pC5). Upon digestion of the plasmid pC5 with Hha I and Dra I a 2.2 kb fragment was released which contained the entire coding sequence of human Pl (from -25 to just before the poly(A) tail). After blunt ending the 5’-end of this fragment, it was subcloned into the Sma I site of the multifunctional vector pGem-7Z (+) (Promega Biological), which is flanked by T7 and SP6 promoters. A clone, PGA8 from which human Pl cDNA is transcribed in the proper orientation by T7 polymerase was chosen for transcription and translation. The deletion of the 5’ leader presequence from PGA-8 was accomplished by Xho I and Ava I double digestion, which removed the initial 107 bases from the Pl-coding sequence leading to initiation at methionine at position 14 of the mature protein (see Fig. 1). The plasmid DNA was prepared by a standard method, and linearized with Hind Ill for in vitro transcription. The expression vector for uncoupling protein (UCP) was pSPTl&UCP which was constructed by cutting pUCP,,,15 (19) with Kpn I and Pst I and inserting the uncoupling protein cDNA into pSPT18 (Pharmacia) for transcription by T7 RNA polymerase. For transcription, the plasmid was cut with Pst I and briefly digested with the Klenow fragment of DNA polymerase. The expression vector for ornithine carbamoyltransferase was pSPO19 (20). 392

Vol.

169, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

General. Procedures for in vitro transcription and translation in a rabbit reticulocyte lysate (Promega Biological) in the presence of [35S]methionine, sodium dodecyl sulfate 12.5% (w/v) polyacrylamide gel electrophoresis and fluorography follow previously described methods (20-21). Import of proteins by isolated rat heart mitochondria also followed previously described procedures (22,23). Incubations were in 150 1.11with 0.5 mg mitochondrial protein/ml for 60 min at 30°C. An uncoupler, carbonyl cyanide mchlorophenylhydrazone (CCCP) at 1.5 mM was used to prevent import. Import within the outer mitochondrial membrane was examined by treating reactions with 15 pg trypsin/pl on ice for 30 min followed by 0.3 mg soybean trypsin inhibitor/ml and 0.2 mM phenylmethylsulfonyl fluoride to stop digestion. RESULTS

AND DISCUSSION

The N-terminal sequence of human hsp60 (or Pl) protein as deduced from the cDNA sequence is shown in Fig. 1. The first 26 amino acids are not present in the mature

protein

mitochondria.

(24) indicating These N-terminal

not contain

any acidic amino

group containing

that they

are removed

during

its transport

residues, which are lacking in the mature protein, do acids and have several basic (viz. Arg) and hydroxyl

(viz. Ser, Thr) amino acids distributed about 4-5 residues apart within hydrophobic region (Fig. 1A). Further, when these residues are

a predominantly arranged along in a helical wheel plot, they form a strongly amphiphilic

=-helix, with all

of the positively charged residues on one face and most of the hydrophobic located on the opposite face (see Fig. lB), which is considered protein translocation into mitochondria (13,17). To investigate and translated

as described in Material and Methods.

Leu Arg t

an essential motif for

The main translation

product of

Upon incubation

Ala

Met

residues

the in vitro import of human hsp60, the protein was transcribed

hsp60 cDNA was a protein of 60-62 kDa (Fig. 2, lane 1).

1

into

19

of this

Phe 0

Leu Pro Thr Val Phe Arg Gln

t

11

Met

Arg Pro Val Ser Arg Val Leu Ala Pro t

21

His Lcu Thr Arg Ala Tyr+t Ala

Helical

wheel

Lys Asp Val

t 31

Lys Phe Gly Ala Asp Ala Arg Ala Leu Met

41

Leu Gin Gly

Val Asp . .. .. . .. .. .. .. . .573 val

A

17

‘Gin 10

B

Fig. 1. Structural features of human P‘1 (hsp60) protein: positively charged residues are indicated by a + and the site of cleavage during maturation is marked by the arrow. (B) Helical wheel plot o f the N-terminal amino acid residues in Pl protein. The The plot was done using HEL\r YHEEL program of PC Gene (Intelligenetics). program plots only 18 amino acids and those showing maximum hydrophobic moment (0.61) are shown. 393

Vol.

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No.

2, 1990

BIOCHEMICAL

protein with rat heart mitochondria

AND

within the matrix since its formation (lane 3) which

that the lower mitochondrial trypsin

prevents

molecular

the precursor

of mitochondria

a cDNA

translation

initiation begins

mitochondria

construct

which

of the uncoupler The inference

form

present

within

that it was not degraded

completely

breaks

was

observed

at methionine

down

upon

or degrades

feature

product

of this cDNA indicates

in the mitochondrial

the

binding of the

import into the matrix compartment.

of Pl protein

precursors of ornithine carbamoyltransferase From a comparison

import

into mitochondria

was associated

was

that in the

with the mitochondria

import of two other proteins, the

(OCT) and uncoupling

protein (UCP) was

of the relative amounts of the 3 proteins present

(lane 1) and that bound to mitochondria

1 Pl

the

No import into

This observation

(Fig. 2, lane 3). To examine this aspect, mitochondrial

during the incubation

and in which

40 (see Fig. 1A).

lanes 2-5).

of CCCP very little of the Pl precursor

also investigated.

import was examined

sequence

of the in vitro translation

amino acid residues

and its subsequent

One surprising

in position

(Fig. 2) (Pl-L

role of the N-terminal

for mitochondrial

lacks the N-terminal

or binding to mitochondria

hsp60 precursor presence

its mature

by our observation (lane 4) which

protein was likely

of proton gradient.

was

of the leader sequence

by making

essential

of Pl

into a

form.

The requirement

construct

form

rapidly converted

in the presence

import by dissipation

mass

COMMUNICATIONS

The mature or imported

was prevented

matrix was supported

treatment

RESEARCH

(lane 2) much of it was

mature form of slightly lower size (~56 kDa). CCCP

BIOPHYSICAL

2

3

4

in the presence of CCCP

5

v-0

P1-L OCT

-

UCP

-4mw-w-

Fig. 2Mitochondrial import of various proteins. Mitochondrial import reactions with various in vitro synthesized [%]methionine-labelled proteins were set up as described under Materials and Methods. Each row represents the experiment with an individual protein (viz. row 1, Pl precursor protein; row 2, Pi protein lacking the leader sequence; row 3, ornithine carbamoyltransferase precursor (00 and row 4, uncoupling protein precursor (UCP)). Lane 1, input protein samples; lanes 2 and 4, samples incubated with mitochondria without trypsin treatment (lane 2) or with trypsin treatment (lane 4). Lanes 3 and 5, samples incubated with mitochondria in the presence of CCCP and either not treated (lane 3) or treated with ttypsin (lane 5). The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and only the relevant portions of the autoradiographs are shown. 394

Vol.

169, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

(lane 3) it is evident that much larger amounts

RESEARCH COMMUNICATIONS

of these latter proteins are bound to

mitochondria as compared to the precursor of Pl protein. Similar results with three proteins have been obtained in two independent experiments. These results indicate that compared to the other two proteins either there are fewer sites for Pi protein on mitochondria PI protein

or the protein is bound less strongly. precursor

mitochondria.

this protein

is very efficiently

imported

into

Previously it had been noted that the binding and import of proteins by

isolated mitochondria of hydrophobic

to mitochondria,

However, despite lower binding of

of Neurospora

crassa was dependent

sequences that followed the presequence

on the presence or absence with hydrophobic

sequences

promoting import (25). It is not known whether this type of effect is occurring with Pl import but the lower binding to mitochondria is striking. In our earlier studies with CHO cells, a number of mutants which exhibited highly specific resistance towards antimitotic drugs electrophoretic alteration in Pl protein (see 26). mitochondrial present.

chaperonin

were found to involve specific How the observed change in this

protein confers resistance to antimitotic

Lastly, although the chaperonin

drugs is unclear at

protein is involved in the mitochondrial

import

and folding of other proteins, its import into mitochondria seems to occur by a similar mechanism to that observed for other proteins. This emphasizes the fact that mitochondrial

biogenesis

depends

upon pre-existing

mitochondria.

ACKNOWLEDGMENT This work was supported of Canada to KBF and RSG.

by research grants from the Medical Research Council

REFERENCES 1. 2. 3. 4. 5. 6. 7.

;: 10. 11.

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