The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix

Abstract

The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino‐terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

“…fntroduction Most nuclear-encoded precursors of mitochondrial proteins contain amino-terminal presequences (for reviews see Pfanner and Neupert, 1987;Nicholson and Neupert, 3988). These presequences are required for the precursors to enter the mitochondrial matrix, where they are proteolytically removed (Hurt et al, 1984;Horwich et al, 1985Horwich et al, , 1986Emr et al, 1986;Keng et al, 1986;Grivell et al, 1987;Vassarotti et al, 1987a). This cleavage is not essential for completing import but is necessary for further assembly of the newly imported polypeptides into functional complexes (Zwizinski and Neupert, 1983;Lewin and Norman, 1983;Ou et al, 1986;Hart1 et al, 1986Hart1 et al, , 1987.…”

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

“…To make pb2(l-167)-DHFR, the DHFR gene from pDS5/2 (Stueber et al, 1984) was excised with BamH1 and HindIII and subcloned into pCLl cut with the same enzymes; the resulting construct encodes the first 167 amino acids of the cytochrome b2 precursor fused to DHFR by the two-residue linker GI. The pb2(l-185)-DHFR was created by cutting plasmid pDS5/2-1-PCOXIV-DHFR (Hurt et al, 1984b) with XhoI, filling in with Klenow, then excising the DHFR gene with HindIII and subcloning it into pCLl cut with EcoRV and HindIII; the resulting construct encodes the first 185 amino acids of the cytochrome b2 precursor fused to DHFR by the five-residue linker SRSGI. Construction of the plasmid-encoding pb2(l-180) has been described (Brunt et al, 1992).…”

Synthesis and Assembly of Mitochondrial Proteins

“…Cytochrome oxidase IV (COX IV) from yeast, an inner membrane protein, is synthesized as a precursor with a 25 amino acid amino-terminal prepiece (Maarse et al, 1984). When progressively truncated parts of the COX IV presequence were fused to DHFR, the first 12 amino acids, but no less, directed DHFR to the mitochondrial matrix both in vivo and in vitro (Hurt et al, 1984b(Hurt et al, , 1985a. When the entire 25 amino acid presequence or the first 22 amino acids were fused to DHFR, proteolytic processing by the matrix peptidase also took place, albeit at an alternative site in the latter case.…”