The yeast nascent polypeptide-associated complex initiates protein targeting to mitochondria in vivo - PubMed (original) (raw)

The yeast nascent polypeptide-associated complex initiates protein targeting to mitochondria in vivo

R George et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The yeast nascent polypeptide-associated complex (NAC) is encoded by two genes, EGD1 and EGD2, and is associated with cytoplasmic ribosomes. Yeast mutants lacking NAC (Deltaegd2) are viable but suffer slight defects in the targeting of nascent polypeptides to several locations including the endoplasmic reticulum and mitochondria. If both NAC and Mft52p are missing from yeast cells, inefficient targeting of mitochondrial precursor proteins leads to defects in both mitochondrial function and morphology. We suggest that NAC provides a ribosomal environment for nascent mitochondrial targeting sequences to achieve secondary structure, thereby enhancing the efficiency of protein targeting.

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Figures

Figure 1

Figure 1

The yeast αNAC homolog Egd2p is associated with free ribosomes. (a) Amino acid sequence alignment of bovine αNAC (11) with the Saccharomyces cerevisiae homolog Egd2p (39). Residues conserved between the two sequences are shaded. (b) Wild-type yeast cells were separated into mitochondria (lane 1), ribosomes (lane 2), and cytosol (lane 3) and were analyzed by SDS/PAGE and immunoblotting against Egd2p, cytochrome _b_2 (cyt _b_2), ribosomal protein L3, and the cytosolic enzyme hexokinase (Hxk). (c) Yeast ribosomes were extracted with high salt, and the extracted proteins were separated by reversed phase HPLC. (d) Mitochondria purified in buffers containing 10 mM EDTA (100 μg of total protein; lane 1) or mitochondria purified in buffer containing 2 mM MgCl2 (100 μg of total protein; lane 2) and free ribosomes (≈2 μg of total protein; lane 3) were analyzed by SDS/PAGE and immunoblotting. (e) The presence of Egd2p (black bars) relative to ribosomal protein L3 (gray bars) was analyzed in samples of free ribosomes (≈2 μg total of protein), mitochondria purified in buffers containing 2 mM MgCl2 (100 μg total of protein), or mitochondria purified in buffer containing 10 mM EDTA (100 μg total of protein). Proteins were separated by SDS/PAGE and blotted to nitrocellulose, and then strips corresponding to the position of Egd2p or L3 were excised, processed, and incubated with _p_-nitrophenolphosphate.

Figure 2

Figure 2

Egd2p is required for efficient delivery of mitochondrial-directed fusion proteins. (a) Mitochondria were prepared from wild-type (lane 1) and Δ_egd2_ (lane 2) cells expressing F1β-LacZ (encoded by the plasmid pCβZ1). (b) Mitochondria were prepared from wild-type (lane 1) and Δ_egd2_ (lane 2) cells expressing CoxIV-DHFR (encoded by the plasmid pKSE). LacZ or DHFR targeted to the mitochondria was measured by immunoblotting after SDS/PAGE. As a control, blots were reprobed with antisera recognizing cytochrome _b_2.

Figure 3

Figure 3

Both Egd2p and Mft52p are involved in mitochondrial biogenesis. (a) Heterozygous (EGD2/Δ_egd2_, MFT1/Δ_mft1_) diploid yeast cells were sporulated, tetrads were dissected, and cells were incubated at 30°C on rich Glc medium for 2 days. (b) Mitochondria were prepared from wild-type (lane 1), Δ_egd2_ (lane 2), Δ_mft1_ (lane 3), and Δ_egd2,_ Δ_mft1_ (lane 4) yeast cells expressing CoxIV-DHFR. DHFR delivered to the mitochondria was measured by immunoblotting after SDS/PAGE. The blots were reprobed with antisera recognizing cytochrome b_2. DHFR delivered to mitochondria was quantitated by densitometry and is expressed as a percentage of the wild-type level of DHFR:cytochrome b_2. (c) Wild-type (diploid) yeast cells were transformed to express CoxIV-GFP. The cells were viewed with Nomarski optics and analyzed by fluorescence microscopy. (d) The homozygous diploid Δ_egd2, Δ_mft1 cells.

Figure 4

Figure 4

Cytoplasmic targeting factors for yeast mitochondrial precursor proteins. For most mitochondrial precursor proteins synthesized in the cytosol, the amino terminus has the potential to form a basic, amphipathic helix. NAC (Egd1p and Egd2p) protects the nascent chain from all cytosolic proteins until 30–50 amino acids have been translated. Subsequently, the targeting sequence becomes accessible to Mft52p and the molecular chaperones Hsp70 (Ssa1p and Ssa2p) and DnaJ (Ydj1p). Other yeast factors, including Mft2p and a homolog of the mammalian protein mitochondrial import stimulating factor (MSF), also may be involved in delivering the precursor protein to the outer membrane translocase (TOM) complex (29) on the mitochondrial surface.

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