Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin is synthesized as a single polypeptide precursor comprising the catalytic A chain and the Gal-binding B chain joined by a 12-amino acid linker propeptide. Upon arrival in the vacuole, the linker is removed. Here, we replicate these events in transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show that the internal linker propeptide is responsible for vacuolar sorting and is sufficient to redirect the ricin heterodimer to the vacuole when fused to the A or the B chain. This internal peptide can also target two different secretory protein reporters to the vacuole. Moreover, mutation of the isoleucine residue within an NPIR-like motif of the propeptide affects vacuolar sorting in proricin and in the reconstituted A-B heterodimer. This is the first reported example of a sequence-specific vacuolar sorting signal located within an internal propeptide.

BP-80, later renamed VSR PS-1 , is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR PS-1 characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR PS-1 together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR PS-1 is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR PS-1 therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR PS-1 receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent. INTRODUCTIONThe plant secretory system is far less easy to study than its yeast counterpart, mainly due to the lack of an equivalent mutant collection. Plant and yeast cells, in opposition to mammalian cells, do not use the mannose 6-phosphatebased lysosomal sorting tag (Kornfeld, 1992); instead, the sorting information is carried by a peptidic sequence. In yeast, the two vacuolar proteins carboxypeptidase Y (CPY; Johnson et al., 1987) and proteinase A (Klionsky and Emr, 1989) both are produced with an N-terminal propeptide responsible for the vacuolar location of the mature protein. Almost 50 mutants defective for vacuolar protein sorting ( vps ) have allowed the identification of proteins involved in the yeast vacuolar sorting pathway. One of these proteins, Vps10p, plays a central role because it is the vacuolar receptor for CPY (Marcusson et al., 1994). Vps10p also is able to send foreign or malfolded proteins to the vacuole for degradation in a process that is believed to be independent of any sorting signal (Hong et al., 1996).In plants, two main types of vacuolar sorting determinants (VSDs) have been well studied and are believed to correspond to two separate vacuolar sorting pathways. The first type could be defined as a sequence-specific VSD (ssVSD) and has been well studied for barley aleurain (Holwerda et al., 1992) and sweet potato sporamin A (Matsuoka and Nakamura, 1991). For these two examples, the VSD is located within an N-terminal propeptide and contains a conserved tetrapeptide NPIR, the Ile being essential (Matsuoka and Nakamura, 1999). The position of this category of VSD seems to be less important than its sequence specificity, as shown with sporamin A (Koide et al., 1997). The second type of VSD is found in C-terminal propeptides (Ct-VSD); it is rather short with no obvious sequence conservation but needs to be accessibl...

SummaryPhaseolin is a vacuolar storage glycoprotein synthesized as a precursor with a short C-terminal propeptide. We have recently shown that deletion of the last four C-terminal amino acids (AFVY, which are part of, or constitute the propeptide) abolishes vacuolar targeting, causing phaseolin to be secreted. Here we provide biochemical and microscopical evidence that the AFVY tetrapeptide, when fused to a secreted version of green fluorescent protein (GFP), inhibits GFP secretion and leads to its accumulation in vacuoles, where it is processed. This demonstrates that the tetrapeptide contains sufficient information for vacuolar sorting.