IN THIS ISSUE

Vacuolar Protein Trafficking and Vesicles: Continuing to Sort It All Out

The array of proteins that any cell will produce (i.e., its "proteome") is, ultimately, a function of its regulated genetic constitution. Once a cell is developmentally committed, however, the correct routing of the proteins that it expresses is just as important to cell function and viability as is the genetic machinery that supports protein expression. Transcription factors, for example, must be routed into the nucleus; glycolytic enzymes must function within the cytosol; certain proteins and hydrolytic enzymes must reach the lytic vacuole (or, equivalently, the lysosome in mammalian cells); membrane proteins must associate correctly with the appropriate organelle; and each cytoplasmic compartment must acquire the proteins and other constituents that render it functional.

Protein trafficking, moreover, is not a unidirectional process. Proteins that have been routed, for instance, as carriers or chaperones of molecular cargo from the endoplasmic reticulum (ER) (i.e. "anterograde" transport), may be recycled back to ER (i.e., "retrograde" transport) for subsequent utility. An additional element of complexity is thus encountered in that the routing machinery must differentiate between molecules appropriate for retrieval and those whose retrieval would merely be counterproductive.

All proteins that are destined for export from the cytosol contain, however transiently, specific peptide sequences that signal them for translocation across or, in the case of membrane proteins, into the cell or organellar membrane. One example of such signal peptides is provided by the hydrophobic N-terminal "preprotein" sequences that promote the cotranslational association of polysomes with the ER. This association, in turn, results in the partial or complete translocation of the nascent protein into the ER lumen, depending on whether or not the protein sequence contains a so-called stop transfer signal, respectively.

To the extent that the ER lumen is topologically equivalent to the cell exterior, protein translocation across the ER membrane amounts to egress from the cell. Indeed, the endomembrane system in general (i.e., the functional integration of the ER, Golgi complex, secretory vesicles, plasma membrane, and vacuolar apparatus) can similarly be equated to the cell exterior. And yet these "exterior" domains are clearly not equivalent: for any given constituent of the endomembrane system, the presence of certain proteins can in fact be taken as diagnostic. Thus, subsequent to the translocation of proteins from the cytosol, the cell retains certain proteins within the ER and specifically routes others to the various domains of the endomembrane system. For any cellular compartment, certain resident proteins will typify the compartment whereas others will be transient (see Vitale and Denecke 1999 , for a review).

Characterization of the biochemical particularities of the various compartments of the endomembrane system continues to be an important task in the cell biology of protein trafficking. Indeed, the existence of such biochemical differences directly reflects the targeted delivery of proteins to specific cellular compartments. Nowhere are the biochemical particularities of protein trafficking more striking than in plant systems, where distinct types of vacuoles can reside within a given cell. And because the delivery of vacuolar proteins is generally mediated by vesicles that envelope the protein cargo at the ER and/or Golgi apparatus, the experimental elucidation of vacuolar protein trafficking thus concerns the char-acterization of intracellular vesicles and how they undergo sorting, targeting, and fusion with other membranous elements. Given that proteins vary so greatly with respect to the ways that they are trafficked—and because each given cell type expresses a definitive proteome—progress in the understanding of protein trafficking generally depends on a variety of approaches in experimental design and a multiplicity of cell types (reviewed in Sanderfoot and Raikhel 1999 ).

Two papers in this issue of THE PLANT CELL address fundamental aspects of protein trafficking. On pages 1509–1524, Hinz et al. report not only on their studies of developing pea cotyledons and the trafficking of legumin, a model storage protein, but also on proteins that are themselves part of the molecular machinery that directly supports protein trafficking. In this way, the article offers the opportunity for readers to review the elegance and complexity of the process as a basic cellular function.

Storage proteins, such as legumin, provide plants with reserves of amino acids that can be readily mobilized for growth processes, and the nutritive value of multiple crop species derives directly from these protein reserves (see Herman and Larkins 1999 ). Storage proteins are housed within the cell in special vacuolar compartments that are distinct from the lytic vacuole, so that storage protein vacuoles (PSVs) represent an additional component of the endomembrane system in some plant cells; as such, PSVs place particular demands upon the protein routing system. To add to this complexity, storage proteins can apparently travel by either of two routes from the ER to the PSV. The first route occurs via the Golgi complex, as is common for the passage of secreted proteins from the ER; specific vesicles subsequently bud off from the Golgi complex to carry storage proteins en route to the PSV. In the "alternate pathway," protein-bearing vesicles bud directly from the ER and are subsequently delivered to the PSV by a Golgi-independent mechanism. Regardless of which route is utilized, the underlying questions are common: How are storage proteins specifically recruited into the vesicles that carry them away from the ER/Golgi apparatus? And how are these vesicles directed to the PSV, rather than to the lytic vacuole or to the plasma membrane?

Previous work has established important themes that continue to guide Hinz et al. in their present contribution. The clathrin protein coat, for instance, is not associated with all vesicles that originate from the Golgi apparatus (Hohl et al. 1996 ). Smooth, electron-dense vesicles that lack the clathrin coat are, at about 100 nm in diameter, significantly larger than their clathrin-coated counterparts. Inasmuch as the clathrin-coated vesicles (CCVs) do not contain detectable prolegumin, the authors surmised that the 100-nm dense vesicles (DVs) are the transporters of the storage proteins. Indeed, CCVs are frequently encountered budding off from DVs and are presumed thereby to remove contaminating proteins that are mistakenly packaged along with storage proteins into the large vesicles.

Because much of the evidence that distinguishes the roles for DVs and CCVs in functional terms is circumstantial, a more systematic consideration of vesicle formation is necessary. As an example, the formation of CCVs from the Golgi complex is dependent on specific adaptor proteins that act to bridge the clathrin coat to integral membrane proteins that extend from the cytoplasmic surface of the vesicles. Within the vesicle lumen, these same integral membrane proteins, in turn, bind the cargo; that is, they function as the receptor proteins that lure protein cargo into the incipiently forming vesicles (see for review Sanderfoot and Raikhel 1999 ). The characterization of the cargo receptors that specifically recruit protein cargo—and thereby effectively establish the identity of the transport vesicle—is thus of prime importance in understanding the cellular panorama of protein trafficking.

In their present report, Hinz et al. probe for a receptor protein involved in the trafficking of vacuolar proteins from the Golgi complex. Specifically, the authors have worked out a method to purify those vesicles that carry prolegumin from developing pea cotyledons. They can then compare the protein profiles of such vesicles with those of CCVs. One of the proteins of interest in these experiments is BP-80, an integral membrane protein previously isolated from pea CCVs for its ability to bind the N-terminal vacuolar protein consensus sequence (Kirsch et al. 1994 ; but see below for an empirical refinement of this characterization). The comparison of BP-80 content is germane because the absence of a clathrin coat from DVs does not preclude the presence of BP-80 or other accessory proteins, such as adaptor proteins or integral membrane proteins like BP-80, that could be involved in vacuolar protein trafficking.

Through a variety of immunological and electron micrographic techniques, the authors conclude that BP-80 is absent from DVs and therefore does not participate in DV-mediated transport. These same techniques go a long way toward corroborating much of the circumstantial evidence that assigns to DVs the role of transporting prolegumin to PSVs. Prolegumin is indeed abundant in the highly purified DVs, as is -TIP (for tonoplast integral protein), which is regarded as a protein marker for the PSV.

A second article in this issue that focuses on protein trafficking within the plant cell also concerns the role of BP-80. On pages 1499–1508, Miller et al. report on the Nicotiana alata proteinase inhibitor (Na-PI), a 40-kD polyprotein that is delivered to the vacuole of stigma cells of N. alata and processed into a series of 6-kD proteinase inhibitors. The authors show that the C-terminal domain of 25 amino acids acts as a preprotein signal sequence that tags the protein for delivery to the vacuole. Indeed, when this C-terminal tag is replaced with a hexahistidine sequence, the resulting protein, rather than reaching the vacuole, is sidetracked into the "default pathway" of protein traffic and is secreted into the medium of cultured transformants. Sorting signals have previously been identified within N-terminal and C-terminal propeptide domains, as well as amid internal sequences that are preserved in the mature protein (see Marty 1999 , for a review). Miller et al. present unexpected cross-linking data, however, in that the sorting receptor for Na-PI is none other than BP-80, heretofore known only to interact with vacuolar proteins characterized by the N-terminal sorting sequence.

Miller et al. further conclude that Na-PI, associating as it does with BP-80, is not carried by DVs to the vacuole. Specifically, they confirm that the DV-specific marker, -TIP, fractionates separately from Na-PI–containing compartments. This conclusion thus corroborates quite nicely the finding of Hinz et al. outlined above, namely, that BP-80 is not associated with DVs. Interestingly, however, a post-Golgi subcellular fraction can be identified that contains Na-PI but that also lacks the vacuole-specific marker -TIP, thereby indicative of a prevacuolar sorting step in the trafficking of Na-PI. Prevacuolar compartments have in fact been identified in both yeast and Arabidopsis through the localization of specific protein markers (Becherer et al. 1996 ; Sanderfoot et al. 1998 ). Although its full significance is not clear, the prevacuolar compartment undoubtedly functions as an additional safeguard to ensure correct delivery of proteins into the central vacuole.

Both of the articles that focus on vacuolar sorting in this issue attest to the complexities of vesicular protein trafficking in plant cells. They both also raise questions that will lead to better characterization of these complexities. In addition to extending our recognition that prevacuolar sorting steps are important for a wider spectrum of plant proteins and tissues than had heretofore been appreciated, the role of the vacuolar protein receptor BP-80 in the trafficking of specific secretory proteins—for example, Na-PI but not prolegumin—has been further clarified.

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Hinz, G., Hillmer, S., Bäumer, M., and Hohl, I. (1999) Vacuolar storage proteins and the putative vacuolar 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].

Hohl, I., Robinson, D.G., Chrispeels, M.J., and Hinz, G. (1996) Transport of storage proteins to the vacuole is mediated by vesicles without a clathrin coat. J. Cell Sci. 109:2539-2550[Abstract].

Herman, E.M., and Larkins, B.A. (1999) Protein storage bodies and vacuoles. Plant Cell 11:601-613[Free Full Text].

Kirsch, T., Paris, N., Butler, J.M., Beevers, L., and Rogers, J.C. (1994) Purification and initial characterization of a potential plant vacuolar targeting receptor. Proc. Natl. Acad. Sci. USA 91:3403-3407[Abstract/Free Full Text].

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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].

Sanderfoot, A.A., Ahmed, S.U., Marty-Mazars, D., Rapoport, I., Kirchhausen, T., Marty, F., and Raikhel, N.V. (1998) A putative vacuolar cargo receptor partially colocalizes with AtPep12p on a prevacuolar compartment in Arabidopsis roots. Proc. Natl. Acad. Sci. USA 95:9920-9925[Abstract/Free Full Text].

Sanderfoot, A.A., and Raikhel, N.V. (1999) The specificity of vesicle trafficking: Coat proteins and SNAREs. Plant Cell 11:629-641[Free Full Text].

Vitale, A., and Denecke, J. (1999) The endoplasmic reticulum—Gateway of the secretory pathway. Plant Cell 11:615-628[Free Full Text].

Related articles in Plant Cell:

Identification and Characterization of a Prevacuolar Compartment in Stigmas of Nicotiana alata

Elizabeth A. Miller, Marcus C. S. Lee, and Marilyn A. Anderson
Plant Cell 1999 11: 1499-1508. [Abstract] [Full Text]  

Vacuolar Storage Proteins and the Putative Vacuolar Sorting Receptor BP-80 Exit the Golgi Apparatus of Developing Pea Cotyledons in Different Transport Vesicles

Giselbert Hinz, Stefan Hillmer, Matthias Bäumer, and Inge Hohl
Plant Cell 1999 11: 1509-1524. [Abstract] [Full Text]  

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