LETTER TO THE EDITOR

Reply: The Role of BP-80 in Sorting to the Vacuole in Stigmas

Jiang and Rogers raise some important issues regarding the complexity of protein traffic to the vacuole in plant cells, a topic that we have studied by examining vacuolar transport of a multidomain precursor protein (Na-PI) that is very highly expressed in stigmas of Nicotiana alata (Atkinson et al. 1993 ). Using this system, we identified a prevacuolar compartment that appeared to be involved in transport of Na-PI to the vacuole. We further determined that within this compartment Na-PI was present in a complex with a homolog of the putative sorting receptor, BP-80 (Miller et al. 1999 ). We also examined the sorting signal required for vacuolar deposition of Na-PI within the more generic system of BY-2 suspension cells. In these cells, deletion of a 25 amino acid C-terminal propeptide domain caused Na-PI to be secreted from the cell rather than accumulated in the vacuole. It is the use of these two different cell types in the characterization of the transport pathway of Na-PI that lead Jiang and Rogers to question some of our interpretations.

We can address some of these questions by clarifying the methods underlying the experiments performed in BY-2 cells. Pulse-chase analysis of the kinetics of transport of Na-PI to the vacuole in stigmas indicated that the transport time of Na-PI, from synthesis to vacuolar deposition, was 2.5 to 3 hours (Elizabeth Miller, unpublished observations). We reasoned that a similar transport time would be required in BY-2 cells and accordingly performed transient expression assays using a 2-hour labeling period. The protein that was analyzed was thus likely to have had time to reach the vacuole (provided that it contained a functional C-terminal sorting signal) but a significant portion would remain unprocessed. Indeed, we were able to identify full-length Na-PI in purified vacuoles. The proteolytic products of Na-PI, 6-kD PIs, were also detected at this time but were not included in the characterization in view of highly homologous endogenous proteins and the proteolytic loss of the epitope tag that had allowed specific detection of the introduced gene. Furthermore, when the BY-2 cells were labeled for longer periods (8 and 24 hours), only the 6-kD products were detected in purified vacuoles, thereby indicating that all of the Na-PI had been directed to a site that contained the appropriate proteolytic enzymes required for its maturation (Elizabeth Miller, unpublished observations). Given that "the vacuolar compartment to which -tonoplast intrinsic protein, a PSV marker, is targeted in tobacco suspension culture cells lacks protease activity" (see Jiang and Rogers' Letter), it seems unlikely that this compartment would be the final destination of Na-PI in BY-2 cells. Thus, we believe that Na-PI is directed to equivalent compartments in both stigmas and BY-2 cells, where the protein is efficiently converted to the mature PI products.

This leads us to the question of the type of vacuole to which Na-PI is directed in BY-2 cells and stigmas. In both cases, the protein reaches a compartment that contains proteolytic activity. Whether this corresponds to a "lytic vacuole" or a vegetative storage vacuole that contains specific proteases responsible for Na-PI maturation remains to be determined and is at issue here. Clearly, the route taken by Na-PI is distinct from the DV pathway. Immunogold electron microscopy of stigma cells shows no evidence for the presence of Na-PI in "dense vesicles" (Elizabeth Miller and Marilyn Anderson, unpublished observations). Furthermore, Na-PI does not form higher order aggregates that are characteristic of proteins trafficked via the DV pathway (Elizabeth Miller and Marcus Lee, unpublished observations; Hinz et al. 1997 ). In addition, the interaction with BP-80 provides further evidence for a transport pathway for Na-PI that is distinct from the DV pathway, a conclusion that was also reached in the accompanying editorial (Smith 1999 ).

Jiang and Rogers would argue that since Na-PI travels predominantly via the lytic pathway, it cannot be sorted via a C-terminal sorting signal and the observation that it interacts with BP-80 is further evidence that additional sorting signals are present. If additional signals are present, however, they do not resemble sorting signals described for other vacuolar proteins that interact with BP-80 (Neuhaus and Rogers 1998 ). Na-PI has neither an N-terminal propeptide nor the NPIR or N(L/I)PS motifs that are necessary for BP-80 binding to proaleurain and 2S albumins, respectively (Neuhaus and Rogers 1998 ). Our transient expression experiments demonstrate that the C-terminal propeptide is absolutely required for vacuolar deposition, and although we cannot rule out the possibility that additional signals contribute to the efficiency of this process, such signals are clearly not sufficient for vacuolar deposition, a diagnostic feature of true sorting signals. Indeed, if Na-PI has a BP-80 binding signal that functions independently of the C-terminal propeptide, deletion of the C-terminal propeptide should not have led to total secretion. Rather, at least some of Na-PI would have been detected in purified vacuoles. Of course, more detailed assessment of the mechanism of interaction between Na-PI and BP-80 will be provided by biochemical characterization of the complex, work that is currently underway. Moreover, given the evidence presented with respect to Na-PI, we must question the validity of equating the storage protein DV pathway with the route taken by proteins with C-terminal propeptide sorting signals. The issue of tissue specificity in vacuolar transport pathways calls for further consideration.

Characterization of the cargo specificity of BP-80 has largely depended on in vitro assays to measure the binding affinity between pea cotyledon BP-80 and synthetic peptides that correspond to sorting signals from vacuolar proteins found in tissues ranging from roots (barley lectin) to seeds (aleurain) and in plants ranging from barley to Brazil nut (Kirsch et al. 1996 ). Such studies may not always reflect the interactions between a vacuolar protein and the sorting receptor(s) in its native tissue. Indeed, given that many plants possess multiple BP-80 genes, we consider it likely that distinct isoforms function in the vacuolar deposition of proteins in a tissue- and/or cargo-specific manner. The route to the plant vacuole may turn out to be even more complex than we currently appreciate.

REFERENCES

Atkinson, A.H., Heath, R.L., Simpson, R.J., Clarke, A.E., and Anderson, M.A. (1993) Proteinase inhibitors in Nicotiana alata are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5:203-213[Abstract].

Hinz, G., Menze, A., Hohl, I., and Vaux, D. (1997) Isolation of prolegumin from developing pea seeds: its binding to endomembranes and assembly into prolegumin hexamers in the protein storage vacuole. J. Exp. Bot. 48:139-149.

Kirsch, T., Saalbach, G., Raikhel, N.V., and Beevers, L. (1996) Interaction of a potential targeting receptor with amino- and carboxyl-terminal targeting determinants. Plant Physiol. 111:469-474[Abstract].

Miller, E.A., Lee, M.C.S., and Anderson, M.A. (1999) Identification of a prevacuolar compartment in stigmas of Nicotiana alata.. Plant Cell 11:1499-1508[Abstract/Free Full Text].

Neuhaus, J.-M., and Rogers, J.C. (1998) Sorting of proteins to vacuoles in plant cells. Plant Mol. Biol. 38:127-144[CrossRef][ISI][Medline].

Smith, H.B. (1999) Vacuolar protein trafficking and vesicles: Continuing to sort it all out. Plant Cell. 11:1377-1379[Free Full Text].

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