Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

Adi Naamati,Neta Regev-Rudzki,Shlomi Galperin,Roland Lill,Ophry Pines

doi:10.1074/jbc.m109.034694

Abstract

In eukaryotes, each subcellular compartment harbors a specific group of proteins that must accomplish specific tasks. Nfs1 is a highly conserved mitochondrial cysteine desulfurase that participates in iron-sulfur cluster assembly as a sulfur donor. Previous genetic studies, in Saccharomyces cerevisiae, have suggested that this protein distributes between the mitochondria and the nucleus with biochemically undetectable amounts in the nucleus (termed "eclipsed distribution"). Here, we provide direct evidence for Nfs1 nuclear localization (in addition to mitochondria) using both alpha-complementation and subcellular fractionation. We also demonstrate that mitochondrial and nuclear Nfs1 are derived from a single translation product. Our data suggest that the Nfs1 distribution mechanism involves at least partial entry of the Nfs1 precursor into mitochondria, and then retrieval of a minor subpopulation (probably by reverse translocation) into the cytosol and then the nucleus. To further elucidate the mechanism of Nfs1 distribution we determined the N-terminal mitochondrial sequence of Nfs1 by Edman degradation. This led to the discovery of a novel mitochondrial processing enzyme, Icp55. This enzyme removes three amino acids from the N terminus of Nfs1 after cleavage by mitochondrial processing peptidase. Intriguingly, Icp55 protease (like its substrate Nfs1) appears to be dual distributed between the nucleus and mitochondria.

Highlights

Eclipsed distribution is a specific case of the dual protein subcellular localization in which one compartment contains the vast majority of a specific protein while a second compartment contains only a minute amount [1,2,3]
We discovered that yeast Nfs1 undergoes two steps of proteolytic processing; first it is cleaved by the mitochondrial processing peptidase (MPP), which removes its mitochondrial targeting sequence (MTS) and it is cleaved by a newly discovered peptidase, designated Icp55, which removes three amino acids from its N terminus
Nfs1 Is Processed by Two Peptidases, MPP and the Novel Icp55— Nfs1 is a highly conserved mitochondrial cysteine desulfurase in all organisms

Summary

EXPERIMENTAL PROCEDURES

Strains—S. cerevisiae strains used were BY4741 (Mata; his3⌬1; leu2⌬0; met15⌬0; ura 3⌬0), W303B1 (Mat␣; leu 112; trp; can100; ura; ade; his3–11,15), ⌬icp (⌬yer078c; derived from a W303B1 strain and was kindly provided by Thomas Langer [13]), and Tet-NFS1 (pNFS1:: kanR-tet07-TATA URA3::CMV-tTA; MATa; his; leu 0; met15– 0) [14]. Plasmids Constructs—NFS1 gene was amplified using yeast genomic DNA as template by PCR using the indicated oligonucleotides (Table 1) and was cloned into vector p425Gal10 [15]. Nuclear ␻ (p␻NLS)LacZ ␻ fragment was amplified from p␻c template using the indicated primers (Table 1); the reverse primer harbors the SV40-T antigen NLS [17], and the PCR product was cloned into pRS426-Met vector. Growth Conditions—Strains harboring the appropriate plasmids were grown overnight at 30 °C in synthetic depleted (SD) medium containing 0.67% (w/v) yeast nitrogen base 2% galactose or 2% glucose (w/v), supplemented with the appropriate amino acids (50 ␮g/ml). Plates containing 2% yeast extract, 1% peptone, and 2% glucose (w/v) (YPD) supplemented with 120 ␮g/ml tetracycline where used for conditional knockout of Nfs for the Tet-NFS1 strain

Metabolic Labeling

Subcellular Fractionation

Mutant name

Protein Purification for Edman Degradation

RESULTS

DISCUSSION