Abstract
Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2a mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells.
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Abbreviations
- AP:
-
Aspartic proteinase
- ER:
-
Endoplasmic reticulum
- PSI:
-
Plant specific insert
- SAPLIP:
-
Saposin-like protein
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Acknowledgments
We are grateful to Jürgen Denecke (University of Leeds, UK) for the gift of anti-calreticulin antibody, to Tony Schaeffner (GSF Research Centre, München, Germany) for the anti-At-α-TIP antibody and to Sandro Vitale for advice on pulse chase and immunoprecipitation. We thank an anonymous reviewer for constructive comments that improved the manuscript. This research was supported by the Portuguese Science and Technology Foundation—Fundação para a Ciência e a Tecnologia (FCT), project POCTI/BME/39765/2001. The corresponding author, Patrícia Duarte, was beneficiary of a PhD grant from FCT.
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Duarte, P., Pissarra, J. & Moore, I. Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway. Planta 227, 1255–1268 (2008). https://doi.org/10.1007/s00425-008-0697-1
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DOI: https://doi.org/10.1007/s00425-008-0697-1