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Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding

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Abstract

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis–trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis–trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)6-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)6-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.

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Abbreviations

Anti-STEP:

Polyclonal antibody preparation directed against castor bean sieve tube exudate proteins

CsA:

Cyclosporin A

FITC:

Fluorescein isothiocyanate

PPIase:

Peptidyl-prolyl cis–trans isomerase

HPLC:

High-pressure liquid chromatography

OG:

Oregon Green

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Acknowledgments

The authors gratefully acknowledge the following people for technical support and analysis: C. Horstmann, Gatersleben, Germany, for separation and sequencing of the cleavage peptides; C. Scholz, Bayreuth, Germany, for the separation of (His)6-tagged RcCYP1 from endogenous bacterial cyclophilin; F. X. Schmid, Bayreuth, for providing the facilities and substrates for kinetic analyses. The Arabidopsis cyclophilin (AtCYP18-3, formerly called ROC1) antibody was generously provided by C. S. Gasser, University of California, Davis. C. Schobert was supported in part by a DAAD fellowship. Support was also provided by a grant from the Deutsche Forschungsgemeinschaft to M.G. and C·S., and by a grant from the U.S. Department of Energy, Division of Energy Biosciences (DE-FG03-94ER20134) to W.J.L.

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Author notes

  1. Beatriz Xoconostle-Cázares

    Present address: Departamento de Biotecnología y Bioingeniería, CINVESTAV IPN. Ave. IPN 2508, Zacatenco, 07360, Mexico DF, Mexico

  2. Christian Schobert

    Present address: Novozymes Biologicals, 111 Kessler Mill Road, Salem, VA, 24153, USA

Authors and Affiliations

  1. Pflanzenphysiologie, Universität Bayreuth, 95440, Bayreuth, Germany

    Maren Gottschalk, Elmar Dolgener, Ewald Komor & Christian Schobert

  2. Section of Plant Biology, College of Biological Sciences, University of California, Davis, CA, 95616, USA

    Beatriz Xoconostle-Cázares, William J. Lucas & Christian Schobert

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  1. Maren Gottschalk
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  2. Elmar Dolgener
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Correspondence to Ewald Komor.

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Gottschalk, M., Dolgener, E., Xoconostle-Cázares, B. et al. Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding. Planta 228, 687–700 (2008). https://doi.org/10.1007/s00425-008-0771-8

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  • DOI: https://doi.org/10.1007/s00425-008-0771-8

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