Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

Abstract

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum

“…An ideal candidate for this type of control is the nascent polypeptideassociated complex (NAC), which competes with the SRP for binding to polypeptides as they emerge from the translating ribosomes and is thought to negatively regulate protein transport to the ER (35,36). Interestingly, NAC has recently been implicated as an enhancer of protein targeting to the mitochondria (37,38). We suggest a working model (Fig.…”

Genome‐wide analysis of mRNAs targeted to yeast mitochondria

“…Although it was later suggested that such locus-specific translation may play a marginal role in the mitochondrial import (Suissa and Schatz, 1982), several recent observations support the idea that a cotranslational process is involved in the mitochondrial import of some proteins (reviewed in Verner, 1993;Lithgow, 2000). The discovery, in mammalian cells, of the nascent polypeptide-associated factor NAC (Wiedmann et al, 1994) and the fact that, in yeast, disruption of either the genes encoding the subunits of the NAC complex or its homologue, the ribosome-associated complex (RAC) (Gautschi et al, 2001), leads to defects in protein targeting to mitochondria (George et al, 1998;Funfschilling and Rospert, 1999) confirm that translation and import are associated.…”

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

“…This binding is independent of signal peptide sequences, and yet NAC cooperates with signal recognition particle (SRP) for correctly targeting and folding of nascent polypeptide in the cotranslational process (Lauring et al, 1995a, b;Powers and Walter, 1996;Moller et al, 1998b). However, yeast NAC homolog (termed as EGD complex) is not prerequisite for growth (Reimann et al, 1999), but is necessary for protein targeting to mitochondria (George et al, 1998;Fünfschilling and Rospert, 1999). In addition, in yeast both subunits including their NAC domains have been shown to be ubiquitinated in a lysine dependent manner (Panasenko et al, 2006(Panasenko et al, , 2009.…”