LETTER TO THE EDITOR

The Role of BP-80 and Homologs in Sorting Proteins to Vacuoles

A recent article (Miller et al. 1999 ) and accompanying editorial (Smith 1999 ) raised issues that are of substantial importance to those of us who putter in the pipes of the secretory pathway. Miller et al. 1999 convincingly showed that tobacco homologs of the vacuolar sorting receptor, BP-80, physically interacted with a ~46-kD Nicotiana alata proteinase inhibitor precursor protein (Na-PI) in stigma cells. This is an important result and the authors deserve congratulations for their fine work. We, however, have serious reservations about conclusions from this experiment. As these conclusions have important ramifications with respect to mechanisms for sorting proteins into the two different Golgi-to-vacuole pathways, it would seem appropriate to bring these issues to the attention of the plant cell biology community.

As was carefully discussed in both the paper and editorial, two pathways that carry soluble proteins from Golgi to vacuoles have been defined. One is followed by the vacuolar sorting receptor BP-80 and involves clathrin coated vesicles (CCVs) for at least a portion of the pathway. All evidence available to date indicates that this pathway leads to a prevacuolar compartment serving the lytic vacuole, defined together as organelles containing an acidic pH and proteases that can process barley proaleurain to mature form (Jiang and Rogers 1998 ). The BP-80 transmembrane domain and cytoplasmic tail direct chimeric reporter proteins to this destination. Indeed, the transmembrane domain alone is sufficient in this regard, and the cytoplasmic tail appears to function in recycling of the protein from prevacuolar compartment back to the Golgi (Jiang and Rogers 1998 ). To the best of our knowledge, the cytoplasmic tail sequences of all BP-80 homologs identified so far have highly conserved motifs thought to function in recruiting the assembly of clathrin coats. These points would argue that different BP-80 homologs are likely to traffic in the same pathway. This hypothesis, of course, remains to be tested and is at issue here.

The second pathway is more complex. In developing pea cotyledon cells, dense vesicles (DVs) clearly define the pathway, and their purification and characterization is a major landmark in plant cell biology. Hinz et al. 1999 and coworkers have shown elegantly that CCVs and DVs differ drastically in their cargo and receptor content. Dense vesicles contain the seed-type storage proteins provicilin and prolegumin, whereas CCVs do not; CCVs contain abundant BP-80, whereas little is found in DVs. DVs traffic to the protein storage vacuole (PSV), whereas, as described above, CCVs are involved in traffic to the lytic prevacuolar compartment.

In cells that do not have a readily identifiable PSV, such as tobacco BY-2 suspension culture cells or leaf mesophyll cells, DVs have not yet been identified morphologically but a functional equivalent of the DV pathway clearly exists. This pathway is defined by its sensitivity to wortmannin and by the nature of sorting determinants that direct soluble proteins into it (reviewed by Neuhaus and Rogers 1998 ). These sorting determinants are usually present in C-terminal propeptides and are characterized by a relative lack of sequence specificity. Barley lectin is a well studied example of a protein that follows this second pathway, and it is directed to vacuoles with the characteristics of PSVs (Jauh et al. 1999 ; Paris et al. 1996 ). Additionally, vacuolar targeting of phaseolin, a seed-type storage protein found in PSVs, requires a classic C-terminal vacuolar sorting determinant (Frigerio et al. 1998 ). Thus, it is likely that the DV pathway and the pathway followed by proteins with the C-terminal determinant are equivalent. In some transgenic tobacco mesophyll cells it is clear that this pathway leads to a vacuole with a neutral pH, separate from the central (presumably lytic) vacuole (Di Sansebastiano et al. 1998 ). Additionally, the vacuolar compartment to which -tonoplast intrinsic protein, a PSV marker, is targeted in tobacco suspension culture cells lacks protease activity for processing barley proaleurain (Jiang and Rogers 1998 ). Thus, what we will call, for simplicity's sake, the DV pathway in suspension culture and mesophyll cells leads to a compartment with little demonstrable lytic function, the PSV equivalent.

To complicate the story further, a different class of storage proteins, vegetative storage proteins, are not directed to seed-type PSVs but rather accumulate in vacuoles that appear to be related to or derived from lytic vacuoles (Jauh et al. 1998 ; Jauh et al. 1999 ). Protease inhibitors, such as sporamin, are vegetative storage proteins; sporamin is thought to be targeted to its vacuole destination by BP-80-type receptors (Neuhaus and Rogers 1998 ). Na-PI is by definition a vegetative storage protein because it is stored in vacuoles in non-seed tissues. Finally, some proteins, such as the barley aspartic proteinase, have determinants that send them through both pathways where they accumulate both in PSVs and lytic vacuoles (Paris et al. 1996 ).

What all this means is that assays for traffic of a protein through the secretory pathway to a vacuole must assay for traffic through both pathways. In the case of Na-PI in stigma cells, where it associates with BP-80 homologs, the predominant vacuole destination is a place where Na-PI is proteolytically processed into small subunits. This observation would be consistent with the above model, where BP-80 traffics to vacuoles that store vegetative storage proteins, that is, vacuoles that may also have lytic functions. However, there is no evidence from that experiment that the tobacco BP-80 homologs bound Na-PI because they specifically recognized the C-terminal propeptide of Na-PI. Maybe they did, but we would argue from the following data that such a possibility is very unlikely.

When Na-PI was expressed in BY-2 tobacco suspension culture cells (Miller et al. 1999 ; Figure 1 therein) the results were essentially opposite those documented in stigma cells. This experiment evaluated only the presence of the ~46-kD precursor form of the protein. When the C-terminal propeptide was present, some ~46-kD precursor was present in vacuoles. (However, as pulse- chase analyses were not performed, it was not possible to assess what fraction—perhaps only a minor fraction—of the initially synthesized molecules remained in ~46-kD form. When the C-terminal propeptide was removed, the ~46-kD form of the protein was secreted and accumulated in the medium. The authors concluded that "in the absence of the CTPP domain, Na-PI does not reach the vacuole" (Miller et al. 1999 ). Because Na-PI goes to a vacuole in stigma cells, and because Na-PI is bound by BP-80 homologs in stigma cells, it was further concluded in the accompanying editorial that BP-80 homologs therefore must interact with the C-terminal propeptide and that they therefore must function in directing proteins into what we call the DV pathway (Smith 1999 ).

These conclusions—particularly, those offered in the editorial—are not warranted by the data. Specifically, the authors did not assess traffic of Na-PI to a lytic vacuole in BY-2 cells. That vacuole should have been a place where the protein would be cut into small pieces, as it is in stigma cells. Presumably because there was a high endogenous background of related small proteins (a warning sign that lytic vacuole processing of Na-PI was likely to occur), the authors could not measure which portion of the newly synthesized precursor was degraded in that manner; the precedent from stigma cells would indicate that this would be the preferred vacuole destination even in BY-2 cells. We argue that the tobacco BP-80 homologs would direct Na-PI to that destination by binding to the protein elsewhere than the C-terminal propeptide. In contrast, preservation of a portion of the precursor form in vacuoles would be fully consistent with traffic via the DV pathway, mediated by the C-terminal propeptide in a BP-80-independent manner, to a PSV equivalent. Thus, Na-PI is likely to carry two different sorting determinants, and is likely to follow two pathways to two separate vacuoles. Until this possibility is assessed in a critical manner, it is premature to conclude that vacuolar sorting receptors of the BP-80 family target Na-PI by binding to its C-terminal propeptide.

REFERENCES

Di Sansebastiano, G.P., Paris, N., Marc-Martin, S., and Neuhaus, J.-M. (1998) Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway. Plant J. 15:449-457[CrossRef][ISI][Medline].

Frigerio, L., de Virgilio, M., Prada, A., Faoro, F., and Vitale, A. (1998) Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. Plant Cell 10:1031-1042[Abstract/Free Full Text].

Hinz, G., Hillmer, S., Bäumer, M., and Hohl, I. (1999) Vacuolar storage proteins and the putative sorting receptor BP-80 exit the Golgi apparatus of developing pea cotyledons in different transport vesicles. Plant Cell 11:1509-1524[Abstract/Free Full Text].

Jauh, G.-Y., Fischer, A.M., Grimes, H.D., Ryan, C.A., and Rogers, J.C. (1998) -Tonoplast intrinsic protein defines unique plant vacuole functions. Proc. Natl. Acad. Sci. USA 95:12995-12999[Abstract/Free Full Text].

Jauh, G.-Y., Phillips, T., and Rogers, J.C. (1999) Tonoplast intrinsic protein isoforms as markers for vacuole functions. Plant Cell 11, in press..

Jiang, L., and Rogers, J.C. (1998) Integral membrane protein sorting to vacuoles in plant cells: Evidence for two pathways. J. Cell Biol. 143:1183-1199[Abstract/Free Full Text].

Miller, E.A., Lee, M.C.S., and Anderson, M.A. (1999) Identification and characterization 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].

Paris, N., Stanley, C.M., Jones, R.L., and Rogers, J.C. (1996) Plant cells contain two functionally distinct vacuolar compartments. Cell 85:563-572[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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