Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate.

Abstract

Abstract.A purified, artificial precursor protein was used as a transport vehicle to test the tolerance of the mitochondrial protein import system. The precursor was a fusion protein consisting of mouse dihydrofolate reductase linked to a yeast mitochondrial presequence; it contained a unique cysteine as its COOH-terminal residue. This COOH-terminal cysteine was covalently coupled to either a stilbene disulfonate derivative or, with the aid of a bifunctional cross-linker, to one of the free amino groups of horse heart cytochrome c.Coupling to horse heart cytochrome c generated a mixture of branched polypeptide chains since this cytochrome lacks a free alpha-amino group. Both adducts were imported and cleaved by isolated yeast mitochondria. The mitochondrial protein import machinery can thus transport more complex structures and even highly charged "membrane-impermeant" organic molecules. This suggests that transport occurs through a hydrophilic environment. large body of evidence suggests that tightly folded proteins cannot be transported across a biological membrane (Zimmermann and Meyer, 1986; Eilers and Schatz, 1988). It is not clear, however, how completely a protein must unfold in order to be accommodated by the translocation machinery. To test the tolerance of this machinery, we have constructed an artificial mitochondrial precursor protein which contains a unique cysteine residue at its COOH terminus. This precursor can be easily and specifically coupled to a variety of molecules and the resuiting adducts can be tested for their ability to import into mitochondria.We show that this artificial precursor can function as the transport vehicle for a "membrane-impermeant" stilbene disulfonate and for cytochrome c that is attached via a branchedchain configuration. This result suggests that import of proteins into mitochondria occurs through a hydrophilic pore which can accommodate structures different from, and more complex than, fully extended linear polypeptide chains. Materials and Methods Construction of the Fusion ProteinThe starting material was the gene-encoding fusion protein 7/188/189 (Vestweber and Schatz, 1988a) inserted into plasmid pDS5/2-1 (Stueber et al., 1984;Hurt et al., 1985). The fusion protein consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor linked to the NH2 terminus of a modified mouse dibydrofolate reductase (DHFR)' whose 1. Abbreviations used in this paper: DHFR, dihydrofolate reductase; MBS, maleimido-benzoyl-N-hydroxysuccinimide ester.COOH terminus had been extended by the addition of glutamine and cystein. The 5' half of the fusion gene was excised as an Eco RI-Sac I fragment and replaced by the corresponding fragment from plasmid pDS5/2-l-pcox IV-DHFR (Hurt et al., 1984); the fusion gene carded by that plasmid encoded the first 22 residues of the cytochrome oxidase subunit IV precursor linked to DHFR. The new fusion gene generated by the exchange of Eco RI-Sac I fragments was excised as an Eco RI-Hind III fragment and inserted into MI3 nap 10. ...

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

“…A logical candidate for an ATP-dependent "import motor" is mitochondrial hsp70 (mhsp70; Kang et al, 1990;Scherer et al, 1990;Manning-Krieg et al, 1991). It is conceivable that the heme-binding domain remains completely folded during translocation, as the mitochondrial import machinery can accommodate structures other than extended polypeptide chains (Vestweber & Schatz, 1988b). However, tightly folded proteins are usually unable to cross the outer membrane (Eilers & Schatz, 1986;Chen & Douglas, 1987;Vestweber & Schatz, 1988a), suggesting that the heme-binding domain unfolds at least partially.…”

Cytochrome c Maturation

“…Ellman's reagent was used according to Riddles et al (18). Thiol modification with 4-acetamido-4Ј-maleimidyl-stilbene-2,2Ј-disulfonate (AMS) (Molecular Probes Inc., Leiden, Netherlands) (19) was carried out by incubating either reduced or oxidized apoprotein (20 M) in freshly prepared AMS solution (15 mM AMS, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% SDS) for 45 min at 37°C with occasional agitation. Buffer exchange to sodium phosphate buffer (50 mM, pH 7.0) was achieved with Centricon Centrifugal filter units (YM-10; Millipore, Bedford, MA) before analysis of the incubated proteins.…”

Acyl Carrier Protein (ACP) Import into Chloroplasts

“…Indeed, pre-holoACP and preapoACP are imported with nearly the same efficiency (Savage and Post-Beittenmiller, 1994). Mitochondria will also import precursor proteins with attached side groups, including stilbene disulfonate and cytochrome c, suggesting that envelope translocation occurs through a hydrophilic pore (Vestweber and Schatz, 1988).…”