Dual Targeting of Spinach Protoporphyrinogen Oxidase II to Mitochondria and Chloroplasts by Alternative Use of Two In-frame Initiation Codons

Abstract

Protoporphyrinogen oxidase (Protox) is the final enzyme in the common pathway of chlorophyll and heme biosynthesis. Two Protox isoenzymes have been described in tobacco, a plastidic and a mitochondrial form. We isolated and sequenced spinach Protox cDNA, which encodes a homolog of tobacco mitochondrial Protox (Protox II). Alignment of the deduced amino acid sequence between Protox II and other tobacco mitochondrial Protox homologs revealed a 26-amino acid N-terminal extension unique to the spinach enzyme. Immunoblot analysis of spinach leaf extract detected two proteins with apparent molecular masses of 57 and 55 kDa in chloroplasts and mitochondria, respectively. In vitro translation experiments indicated that two translation products (59 and 55 kDa) are produced from Protox II mRNA, using two in-frame initiation codons. Transport experiments using green fluorescent proteinfused Protox II suggested that the larger and smaller translation products (Protox IIL and IIS) target exclusively to chloroplasts and mitochondria, respectively.

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

“…(24) Alternative TISs and leaky scanning are responsible for the synthesis of plastidic and cytoplasmic/ nuclear isoforms of tRNA ligase of Arabidopsis thaliana and Oryza sativa, plastidic and mitochondrial isoforms of spinach protoporphyrinogen oxidase and arabidopsis DNA polymerase. (15,25,26) The mRNA of a human spastin gene contains two alternative translation start sites and produces two protein isoforms, 68 and 60 kDa, respectively. Both isoforms are imported into the nucleus, but the 68-kDa isoform contains two nuclear export signals that efficiently drive its export to the cytoplasm.…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…Proteomic analysis identified both PPO isoforms in plastids membranes (Joyard et al 2009); PPO1 (At4g01690) is associated with both envelope and thylakoid membranes, whereas PPO2 (At5g14220) is restricted to the envelope membrane. On the other hand, dual localization of PPO in both plastids and mitochondria is proposed in tobacco (Lermontova et al 1997) and spinach (Watanabe et al 2001). In spinach, the PPO2 isoform has translational variants and the larger (57 kDa) and smaller (55 kDa) forms are targeted into plastids and mitochondria, respectively (Watanabe et al 2001).…”

Resistance to Protoporphyrinogen Oxidase-inhibiting Compound S23142 from Overproduction of Mitochondrial Protoporphyrinogen Oxidase by Gene Amplification in Photomixotrophic Tobacco Cells

“…8) The tobacco (Nicotiana tabacum) used in this study is an allotetraploid derived from ancestors of the modern diploids, Nicotiana sylvestris and Nicotiana tomentosiformis.…”

“…The proteins were separated by SDS-PAGE (12.5z, v W v) as described by Laemmli 25) and electrophoretically transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia) with a Bio-Rad semidry blotter (Bio-Rad, Hercules, CA, U.S.A.). The membranes were incubated with the primary anti-Protox I antibody (1:2000 dilution), 7) anti-Protox II antibody (1:1000 dilution), 8) or anti-cytochrome c antibody (clone no. 7H8.2C12; Pharmingen; 1:250 dilution) for 1 h at room temperature and subsequently incubated with their respective secondary antibodies conjugated to horseradish peroxidase.…”

“…7,8) In tobacco (Nicotiana tabacum cv. Samsun NN), two cDNAs have been identiˆed to encode the two Protox proteins, PPX-I and PPX-II, that were only 27.3z similar.…”