Dual targeting and function of a protease in mitochondria and chloroplasts

Abstract

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP-GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.

Figure 1

Targeting of the green fluorescent protein fusions to tobacco protoplasts. (A) The amino-terminal 125 amino-acid residues of the Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) precursor protein. The predicted targeting sequence is shown in blue and two potential initiation codons are in red. (B) Schematic presentation of the three green fluorescent protein (GFP) fusion constructs used in the transient expression experiments. (C) Transient expression of the GFP fusion constructs in Nicotiana tabacum protoplasts: F1β–GFP (Ca–Cd), soluble GFP (Ce–Ch), Zn-MP–GFP (Ci–Cl), Δ1–28Zn-MP–GFP (Cm–Cp) and [M29L]Zn-MP–GFP (Cq–Ct), as described in Methods. The GFP column shows the signal detected in the green channel; the Mitotracker column shows the signal detected in the red channel; the GFP + Mitotracker column corresponds to the merging of the two previous columns, in which yellow represents the superposition of green and red; and the GFP + chlorophyll column corresponds to the merging of the green channel and the chlorophyll signal detected in the far-red channel. Scale bars, 10 μm.

Figure 2

Targeting of the green fluorescent protein fusions to tobacco-leaf epidermal cells. Agrobacterium-mediated transient expression of the green fluorescent protein (GFP) fusion constructs into the Nicotiana tabaccum leaves: F1β–GFP (A,B), soluble GFP (C,D), Zn-MP–GFP (E,F), Δ1–28Zn-MP–GFP (G,H) and [M29L]Zn-MP–GFP (I,J) as described in Methods. Scale bars, 10 μm.

Figure 3

In vitro import of the Arabidopsis thaliana zinc-metalloprotease into mitochondria and chloroplasts. (A) The indicated precursors were incubated with mitochondria as described in Methods. Proteinase K (15 μg ml⁻¹) was added after import or 1 μM valinomycin was added before import, as indicated. (B) The indicated precursors were incubated with chloroplasts. Thermolysin (5 μg ml⁻¹) was added after import where indicated. (C) In vitro import of the Zn-MP precursor into the dual import system. The Zn-MP precursor was incubated with mitochondria and chloroplasts together. Mitochondria and chloroplasts were re-isolated on a 4% Percoll gradient after import, as described in Methods. Zn-MP, zinc-metalloprotease; Δ1-28Zn-MP, truncated Zn-MP presequence; [M29L]Zn-MP, mutant Zn-MP precursor. m, mature form; p, precursor.

Figure 4

Dual localization of the zinc-metalloprotease in spinach. Recombinant Arabidopsis thaliana zinc-metalloprotease (AtmZn-MP), spinach mitochondria (Mit) and chloroplasts (Chl) were immunodecorated with antibodies raised against the carboxy-terminal part of the AtmZn-MP, as described in Methods. m, mature form.

Figure 5

Dual proteolytic activity of the recombinant Arabidopsis thaliana zinc-metalloprotease. Lanes 1 and 4, the mitochondrial presequence peptide, N_5.7pF₁β(2–54)-hsl and the chloroplastic transit peptide, SStpPs alone. N_5.7pF₁β(2–54)-hsl and SStpPs peptides were either incubated (lanes 2, 3, 5 and 6) or not (lanes 1 and 4) with recombinant Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of o-phenanthroline, as described in Methods. The sequence of both peptides is depicted at the top.