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Plant and Cell Physiology Advance Access originally published online on April 1, 2008
Plant and Cell Physiology 2008 49(5):751-766; doi:10.1093/pcp/pcn051
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Tomato Rab11a Characterization Evidenced a Difference Between SYP121-Dependent and SYP122-Dependent exocytosis

Reiaz Ul Rehman1,4, Egidio Stigliano1,4,5, Grantley W. Lycett2, Liliane Sticher3, Francesca Sbano1, Marianna Faraco1, Giuseppe Dalessandro1 and Gian-Pietro Di Sansebastiano1,*

1Di.S.Te.B.A., Università del Salento, via prov. Lecce-Monteroni, 73100 Lecce, Italy
2Plant Sciences Division, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK
3Department of Biology, Plant Biology, University of Fribourg, ch. Du Musée 10, CH-1700 Fribourg, Switzerland

*Corresponding author: E-mail, gp.disansebastiano{at}unile.it; Fax, +39–0832298858.


  Abstract

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat β-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory events.

Keywords: Dominant negative mutants - Exocytosis —Plasma membrane - Rab11 - Syntaxin

Abbreviations: ANOVA, analysis of variance; BFA, brefeldin A; CA, constitutively active; DN, dominant negative; EE, early endosome; ER, endoplasmic reticulum; GFP, green fluorescent protein; PEG, polyethylene glycol; PM, plasma membrane; PVC, pre-vacuolar compartment; RFP, red fluorescent protein; SecRGUS, secreted rat β-glucuronidase; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TBS, Tris-buffered saline; TGN, trans-Golgi network; TMD, transmembrane domain; YFP, yellow fluorescent protein.

4These authors contributed equally to this work.

5Present address: Institute of Botany, University of Neuchatel, Rue E. Argand 9, CH-2007 Neuchatel, Switzerland.

(Received December 3, 2007; Accepted March 21, 2008)
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