Poster: Protein targeting & vesicular trafficking

Abs # 645: Intracellular localization and targeting of a programmed cell death-associated broad bean cysteine proteinase, vfcyspro.

Presenter:	Trobacher, Chris P., ctrobacher@hotmail.com
Authors	Trobacher, Chris P. (A)   Mullen, Robert T. (A)   Greenwood, John S. (A)
Affiliations:	(A): Department of Botany, University Of Guelph

Proteases are involved in programmed cell death (PCD) in both animals and plants. In plants cysteine proteinases are emerging as enzymes common to PCD across species. Many of these proteinases possess a carboxy-terminal endoplasmic reticulum (ER) retention signal and localize to precursor protease vesicles (PPVs). During PCD the PPVs promote cellular degradation presumably by fusing with the vacuole, releasing the proteases into the vacuolar lumen. The plant cell vacuole then participates in PCD by engulfing cytoplasm and organelles for digestion prior to cell death, or by bursting and releasing lytic enzymes into the cytoplasm where they act to dismantle the cell. A cysteine proteinase, VFCYSPRO, isolated from broad bean (Vicia faba L.) is expressed in dying tissues and does not possess a carboxy-terminal ER retention signal which begs the question of its intracellular localization. In this study we use laser-scanning confocal microscopy on Vicia faba L. protoplasts transiently expressing both a yellow fluorescent protein-tagged VFCYSPRO and a vacuolar cyan fluorescent protein. The results demonstrate that VFCYSPRO is a bona fide vacuolar enzyme and that it does not localize to structures resembling PPVs. This localization supports the hypothesis that VFCYSPRO is involved in PCD, and is unique because the enzyme lacks the NPIR consensus sequence found in other amino-terminal propeptide vacuolar sorting determinants (VSDs) identified to date.