Progress 03/01/03 to 02/28/06
Outputs
Retired nothing to report
Impacts
Retired nothing to report.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
There were two major accomplishments this past year. (1) We identified the sequence PIEPPPHH in the cytoplasmic tail of a reporter protein that causes the protein to traffic from the endoplasmic reticulum (ER) to the prevacuolar compartment for the protein storage vacuole (PSV). We then identified a protein, termed SRC2, that interacts with the sequence. SRC2 is present in the ER and then moves with the reporter protein to the prevacuolar organelle, and then to the PSV. Both SRC2 and the reporter are internalized within the prevacuolar organelle as it buds from the ER. Overexpression of SRC2 causes the reporter and SRC2 to be retained in the ER, which then appears dilated. Thus SRC2 appears to play a major role in the process by which budding of the prevacuolar organelle occurs, and probably is responsible for causing the reporter protein to be incorporated into the organelle. (2) RMR proteins are receptor like proteins that move from ER to Golgi to PSV in plant
cells. We have used a recombinant RMR protein synthesized in and secreted from insect cells to study its ability to bind to potential ligand. The protein binds specifically and with very high affinity to C-terminal propeptides that are known to be targeting sequences that direct the protein to which they are attached to the PSV. For binding to occur the peptide sequence must be attached to a rigid matrix and must be presented with a free C-terminus. RMR proteins co-immunoprecipitate with a reporter protein carrying the tobacco chitinase C-terminal propeptide, but not with the same reporter lacking the propeptide. When expressed in protoplasts, the RMR protein and the tobacco chitinase propeptide reporter colocalize in prevacuolar organelles. These results are consistent with RMR proteins serving as sorting receptors for the PSV pathway.
Impacts
Our studies provide more detailed information on mechanisms that direct proteins to a storage destination. The results may be useful in biotechnology where it is desired to store a recombinant protein in a place where it will be protected from degradation.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The central accomplishment of the laboratory this past year was to establish that protein storage vacuoles in plant seeds are compound organelles. In other words, they are surrounded by a membrane that defines the organelle. Inside, however, are two compartments. The "soup" represents a storage compartment where storage proteins are packaged. There is also an internal vacuole surrounded by a single membrane. This internal vacuole-within-a-vacuole, the globoid compartment, contains crystals of phytic acid as well as hydrolytic enzymes that are characteristic of a lytic or digestive vacuole. The globoid compartment membrane contains integral membrane proteins consistent with those of a lytic vacuole: gamma tonoplast intrinsic protein and the proton pump, vacuolar pyrophosphatase. We speculate that, as a seed takes up water prior to germination, the membrane around the globoid compartment would be disrupted, allowing the internal enzymes access to the storage compartment
to allow rapid initiation of storage protein breakdown. We have subsequently pointed out that, although storage functions are most prominent in plant cells, mammalian cells also have the equivalent of this compound vacuole. In both plant and mammalian cells, there are two separate pathways that carry proteins and membrane to the two compartments.
Impacts
Our observations raise questions about how a single membrane-bound internal vacuole can be formed within a vacuole that require new explanations for membrane partitioning and delivery of vesicle contents.
Publications
- Jiang, L. and Rogers, J.C. 2002 Sorting of Lytic Enzymes in the Plant Golgi Apparatus. In 'Annual Plant Reviews, Vol. 7: The Plant Golgi Apparatus' (Robinson, D.G., ed.), Sheffield Academic Press, Sheffield, pp. 114-140.
- Jiang, K., Erickson, A.H. and Rogers, J.C. 2002 Multivesicular Bodies: A Mechanism to Package Lytic and Storage Functions in One Organelle? Trends Cell Biol. 12:362-367.
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Progress 01/01/01 to 12/31/01
Outputs
We have proven that protein storage vacuoles have two compartments, one for storage products, and one, the globoid cavity, surrounded by its own membrane and representing an internal vacuole that contains lytic enzymes.
Impacts
This will have profound implications for understanding how storage proteins in seeds are protected during seed development, but are rapidly degrading during germination. Our model suggests that the lytic components of the globoid are likely to have very rapid access to the storage components during germination, possibly by disruption of the globoid membrane as the seed dessicates.
Publications
- Jiang, L. and J.C. Rogers. 2001. Compartmentation of Proteins in the Protein Storage Vacuole, A Compound Organelle in Plant Cells. Adv. Bot. Res. 35:140-170.
- Jiang, L., R.C. Phillips, C.A. Hamm, Y.M. Drozdowicz, P.A. Rea, M. Maeshima, S.W. Rogers, and J.C. Rogers. 2001. The Protein Storage Vacuole: A Unique Compound Organelle. J. Cell Biol. 155:991-1002.
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Progress 01/01/00 to 12/31/00
Outputs
We have characterized the structural components of a plant vacuolar sorting receptor that are necessary for ligand binding. We have developed a recombinant system for expressing large amounts of the protein and are working to crystallize it and solve its three-dimensional structure. We have demonstrated that the protein storage vacuole is a compound organelle where the storage products are separate from an internal, membrane-bound compartment that represents a lytic vacuole. We have identified and characterized a cytoplasmic multivesicular body that serves as the prevacuolar organelle for the PSV. We have identified a receptor-like protein that traffics from ER to Golgi to this prevacuolar organelle that may function in sorting storage proteins into the pathway.
Impacts
In plant biotechnology, in order to make and accumulate many compounds of interest we will have to provide the proper place to store them. Most of them are stored in vacuoles and nobody understands what goes into making the right vacuolar environment. An understanding of how plant cells generate and maintain specialized storage vacuoles will make that goal possible.
Publications
- Cao, X., Rogers, S.W., Butler, J., Beevers, L. and Rogers, J.C. 2000. Structural Requirements for Ligand Binding by a Probable Plant Vacuolar Sorting Receptor. Plant Cell 12:493-506.
- Jiang, L., Phillips, T.C., Rogers, S.W. and Rogers, J.C. 2000. Biogenesis of the Protein Storage Vacuole Crystalloid. J. Cell Biol. 150:755-769.
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Progress 01/01/99 to 12/31/99
Outputs
Expressed multiple forms of the vacuolar sorting receptor in insect cells and determined structural requirements for ligand binding; defined functionally distinct vacuoles by the composition of their membranes; determined what amino acid sequences were needed to direct integral membrane proteins to different vacuoles; characterized the substrate preferences for the plant golgi Kex2p protease; cloned and characterized a unique RNase expressed in barley aleurone cells under GA-regulation.
Impacts
(N/A)
Publications
- Jauh, G.-Y., Phillips, T.E. and Rogers, J.C. 1999 Tonoplast Intrinsic Protein Isoforms as Markers for Vacuolar Functions. Plant Cell 11:1867-1882.
- Jiang, L. and Rogers, J.C. 1999 Functional Analysis of a Golgi-localized Kex2p-like Protease in Tobacco Suspension Culture Cells. Plant J. 18:23-32.
- Jiang, L. and Rogers, J.C. 1999 Sorting of Membrane Proteins to Vacuoles in Plant Cells. Plant Sci. 146:55-67.
- Jiang, L. and Rogers, J.C. 1999 The Role of BP-80 and Homologs in Sorting Proteins to Vacuoles. Plant Cell 11:2069-2071.
- Rogers, J.C. 1999 A Mystery Within an Enigma. Book Review: The Plant Vacuole. Trends Plant Sci. 4:21.
- Rogers, S.W. and Rogers, J.C. 1999 Cloning and Characterization of a Gibberellin-induced RNase Expressed in Barley Aleurone Cells. Plant Physiol. 119:1457-1464.
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Progress 01/01/98 to 12/31/98
Outputs
We have used antibodies specific for four different tonoplast intrinsic proteins (TIPs) to determine the association of different TIP isoforms with functionally distinct vacuoles. These results have extended the description of functionally distinct vacuoles as follows: delta-TIP is specifically associated with storage vacuoles; together with alpha-TIP it defines protein storage vacuoles that store seed-type storage proteins in protein bodies, while alone or together with gamma-TIP it defines vacuoles that store pigments and vegetative storage proteins. gamma-TIP alone defines lytic vacuoles with an acidic pH and active proteases, while alpha-TIP alone defines autophagic vacuoles. We have used a chimeric reporter protein expressed in tobacco suspension culture protoplasts to study traffic of integral membrane proteins to different vacuoles. The default pathway for integral membrane proteins is from ER to Golgi to the lytic vacuole. In contrast, the carboxyl-terminal
cytoplasmic tail of alpha-TIP, when attached to the reporter protein, directs the protein into a novel pathway directly from the ER to the protein storage vacuole equivalent in the protoplasts.
Impacts
(N/A)
Publications
- Rogers, J.C. 1998. Aleurain. In "Handbook of Proteolytic Enzymes" (Barrett, A.J., Rawlings, N. and Woessner, F., eds.), Academic Press, London, pp. 583-585.
- Rogers, J.C. 1998. Compartmentation of plant cell proteins in separate lytic and protein storage vacuoles. J. Plant Physiol. 152:653-658.
- Neuhaus, J.-M. and Rogers, J.C. 1998. Sorting of proteins to vacuoles in plant cells. Plant Mol. Biol. 38:127-144.
- Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. HRT, a novel zinc finger, transcriptional repressor from barley. J. Biol. Chem. 273:23313-23320.
- 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.
- Jauh, G.-Y., Fischer, A.M., Grimes, H.D., Ryan, C.A. and Rogers, J.C. 1998. delta-Tonoplast intrinsic protein defines unique plant vacuole functions. Proc. Natl. Acad. Sci. USA 95:12995-12999.
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