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C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

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Summary

The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 μm diameter), a characteristic of so-called “unspecialized” peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to β-ATPase and stearoyl-ACP Δ 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.

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Abbreviations

CAT:

chloramphenicol acetyltransferase

CHO:

cells Chinese hamster ovary cells

DAB:

3,3′-diaminobenzidine

GUS:

β-glucuronidase

ICL:

isocitrate lyase

KSRM:

lysine-serine-arginine-methionine

MS:

malate synthase

PBS:

phosphate-buffered saline

PTS:

peroxisome targeting signal

SKL:

serine-lysine-leucine

tobacco BY-2:

Bright Yellow-2

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Authors and Affiliations

  1. Department of Botany, Arizona State University, Box 871601, 85287-1601, Tempe, AZ, USA

    R. N. Trelease, M. S. Lee, A. Banjoko & J. Dunkelmann

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  1. R. N. Trelease
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  2. M. S. Lee
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  3. A. Banjoko
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  4. J. Dunkelmann
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Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology

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Trelease, R.N., Lee, M.S., Banjoko, A. et al. C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells. Protoplasma 195, 156–167 (1996). https://doi.org/10.1007/BF01279194

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