Folding of Fumarase during Mitochondrial Import Determines its Dual Targeting in Yeast

Ehud Sass,Sharon Karniely,Ophry Pines

doi:10.1074/jbc.m302344200

Ehud Sass, Sharon Karniely + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m302344200

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Abstract

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Thus, fumarase processed in mitochondria returns to the cytosol. In the current work, we (i) generated mutations throughout the coding sequence which resulted in fumarases with altered conformations that are targeted to mitochondria but have lost their ability to be distributed; (ii) showed by mass spectrometry that mature cytosolic and mitochondrial fumarase isoenzymes are identical; and (iii) showed that hsp70 chaperones in the cytosol (Ssa) and mitochondria (Ssc1) can affect fumarase distribution. The results are discussed in light of our model of targeting and distribution, which suggests that rapid folding of fumarase into an import-incompetent state provides the driving force for retrograde movement of the processed protein back to the cytosol through the translocation pore.

Highlights

Of significant tertiary structure formation, yet once folded in the cytosol, it is in an enzymatically active state and not competent for import
We (i) generated mutations throughout the coding sequence which resulted in fumarases with altered conformations that are targeted to mitochondria but have lost their ability to be distributed; (ii) showed by mass spectrometry that mature cytosolic and mitochondrial fumarase isoenzymes are identical; and (iii) showed that hsp70 chaperones in the cytosol (Ssa) and mitochondria (Ssc1) can affect fumarase distribution
We show that deletions within each one of these domains abolished dual targeting, with essentially all of the fumarase being targeted to mitochondria (Fig. 1A, ⌬33–91, ⌬256 –350, and ⌬421– 488)

Summary

EXPERIMENTAL PROCEDURES

Strains and Plasmids—The S. cerevisiae strains used were DMM1– 15A (leu, ura, ade, his5) [1], YPH499 (ura, lys, ade, trp, his, leu2) [10], E290 (his, trp1) [11], MW141 (leu, ura, lys, trp, his, ssa1::HIS3, ssa2::LEU2, ssa4::URA3, pGAL1-SSA1 [12]), W303–1A (leu, his, trp, ura, ade, can1), and PK81 (␣-ade, lys, ura, leu, trp, ssc1–2(LEU2)) [13]. P⌬B contains a deletion of the DNA sequence encoding amino acids 335–350; we used primers 5Ј-TTGGGTCGACCTGCAGC-3Ј and 5Ј-GGGGTTAACTTCATGGTAGCCGCAACG-3Ј and cloned the PCR product (on an FUM1 template) into the SalI and HpaI sites of pFT2. The labeled cells were collected by centrifugation and resuspended in TE buffer (pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride, broken with glass beads, and centrifuged to obtain the supernatant fraction. The cross-linker was incubated at 4 °C with purified mitochondria prepared by standard subcellular fractionation, as described previously [9], and the reaction was stopped by using 40 mM ammonium chloride. Enzyme Activities—Induced cultures (in galactose) were harvested and resuspended in TE buffer (pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride, broken with glass beads, and centrifuged to obtain the supernatant fraction. Samples were analyzed on 10% SDSPAGE, electro-blotted, and probed with fumarase antiserum

RESULTS

Chromosomal knockout

Amino acid sequence

DISCUSSION