Nuclear Localization of Yeast Nfs1p Is Required for Cell Survival

Yumi Nakai,Masato Nakai,Hideyuki Hayashi,Hiroyuki Kagamiyama

doi:10.1074/jbc.m007878200

Abstract

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.

Highlights

The NifS protein is found in various organisms and is involved in iron-sulfur protein biosynthesis
We identified a potential nuclear localization signal (NLS) sequence in the mature domain of yeast Nfs1p and showed in vivo that this sequence was crucial for the physiological function of extramitochondrial Nfs1p
Most bacterial NifS proteins, including A. vinelandii NifS, had somewhat diverse sequences in the corresponding region, including one neutral residue instead of the basic residue in the third or the fourth position of the sequence (Fig. 1B). Both the E. coli and A. vinelandii IscS proteins possess the RRKPR sequence, which is similar to the NLS-like sequence found in eukaryotic Nfs1 proteins (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis of the NFS1 Gene and Plasmid Construction—A 5Јterminal 500-base pair fragment of the NFS1 gene was amplified by PCR from genomic DNA isolated from the wild-type strain W303–1B [20] It was cloned into the URA3-containing vector pYX013 (Ingenius) downstream of the GAL1 promoter. To construct a plasmid that constitutively expresses the wild-type Nfs1p, the entire NFS1-coding region was amplified by PCR and subcloned into a CEN4-based TRP1-containing plasmid pTT-GAP [21] so the inserted NFS1 could be expressed under the constitutive glycelaldehyde-3-phosphate dehydrogenase (GAP1) promoter. Met104 and the Met118 in yeast Nfs1p were replaced with alanine by site-directed mutagenesis, and the resulting mutant NFS1-h6 genes

This study

RESULTS

DISCUSSION