Plant Molecular Biology 51: 631–641, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 631 The position of the proricin vacuolar targeting signal is functionally important Nicholas A. Jolliffe, Aldo Ceriotti † , Lorenzo Frigerio and Lynne M. Roberts ∗ Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK and † Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, via Bassini 15, 20133 Milano, Italy. ( ∗ author for correspondence; E-mail: lroberts@bio.warwick.ac.uk) Received 2 April 2002; accepted in revised form 27 June 2002 Key words: propeptide, protein sorting, ricin, sequence-specific vacuolar sorting signal, vacuole Abstract Ricin is synthesised as an ER-targeted precursor containing an enzymatic A chain and a galactose-binding B chain separated by a 12-amino acid linker propeptide. This internal propeptide is known to contain a sequence- specific vacuolar sorting signal whose functionality depends on the presence of an isoleucine residue. Conversion of this isoleucine to glycine completely abolished vacuolar targeting of proricin and led to its secretion. However, when this mutated signal was positioned at the C-terminus of a normally secreted reporter, vacuolar targeting of a significant fraction still occurred. Likewise, when the corrupted linker was C-terminally exposed within its natural context following the mature ricin A chain, and then co-expressed with ricin B chain, toxin heterodimers were still partially transported to tobacco cell vacuoles. By contrast, when placed at the N-terminus of the secreted reporter, or at the N-terminus of ricin B chain for co-expression with ricin A chain, the propeptide behaved most strikingly as a sequence-specific vacuolar targeting signal that, when mutated, resulted in complete secretion of the proteins. It would appear that the position of the linker peptide influences the specificity of its vacuolar targeting function. Introduction At least two different types of vacuoles can co-exist within the same plant cell (Di Sansebastiano et al., 1998; Paris et al., 1996), and these may be dis- tinguished biochemically by the presence of specific marker proteins (Vitale and Raikhel, 1999). Lumi- nal proteins therefore require specific sorting signals to reach the correct type of vacuole from their origi- nal site of deposition and folding in the endoplasmic reticulum (ER). These signals fall into three classes. Sequence-specific vacuolar sorting signals (ssVSS) contain a conserved sequence, NPIR or similar, which does not tolerate major perturbation. These usually occur as propeptides at the N-terminus of proteins targeted to the lytic vacuoles (LV), such as spo- ramin (Matsuoka and Nakamura, 1999) and barley aleurain (Holwerda et al., 1992). A second group constitutes the C-terminal signals (ctVSS) that reside within propeptides on proteins destined for the protein storage vacuoles (PSV), including barley lectin (Dom- browski et al., 1993), bean phaseolin (Frigerio et al., 1998a), brazil nut 2S albumin (Saalbach et al., 1996) and tobacco chitinase A (Neuhaus et al., 1994). These signals have no standard length or sequence homol- ogy, apparently sharing only a general hydrophobicity, and the need to be C-terminally exposed in order to function (Matsuoka and Neuhaus, 1999; Vitale and Raikhel, 1999). Known signals present within N- or C-terminal propeptides are removed once the protein has reached the vacuoles. A third group of targeting signals are the ill-defined physical structure signals (psVSS) that are believed to occur within the mature folded domain of proteins such as phytohemagglu- tinin (von Schaewen and Chrispeels, 1993). The signal present in the saposin domain of phytepsin may also fall in this last category (Törmäkangas et al., 2001). Ricin is an example of a vacuolar protein whose VSS is not typical of any of the categories described above. It is a dimeric cytotoxic lectin that normally