Progress 07/01/00 to 09/30/04
Outputs
Research concentrated on protein dynamics within the secretory pathway and the assembly of the central organelle within that pathway, the Golgi apparatus. All experiments were done with HeLa cells, a human cell line. In brief, two central discoveries were made. 1) The resident proteins of the Golgi apparatus such as Golgi glycosyltransferases cycle continuously to the endoplasmic reticulum by two mechanisms. A constitutive mechanism and another, induced by apparent up-regulation of rab proteins, small GTPases of the monomeric G protein superfamily. Rab proteins, in particular rab6, were found to regulate Golgi apparatus redistribution in response to hypotonic stress. This finding places Golgi apparatus protein cycling into the context of a regulated cellular response. 2) The kinetics of Golgi apparatus assembly was "sandwich-like" with the medial Golgi components assembling more slowly into a compact, juxtanuclear structure than cis (entry side) or trans (exit side)
components. This finding was surprising and not predicted by models of Golgi function in secretion such as cisternal maturation or a stable compartment model. These discoveries relate to understanding how the Golgi apparatus reassembles at the end of mitosis in the cell cycle.
Impacts
Discovery from this research has altered our understanding of how cells assemble organelles involved in secreting or moving materials out of the cell. This assembly process is called retrograde cycling within the secretory pathway provides a novel pathway for drug delivery to the cell interior.
Publications
- Storrie, B. 2005. Making ends meet: Maintenance of Golgi apparatus structure in the face of continuous protein recycling to the ER. Int. Rev. Cytol. (In press).
- Kasap, M., S. Thomas, E. Danaher, V. Holton, and B. Storrie. 2004. Dynamic nucleation of golgi apparatus assembly from the endoplasmic reticulum in interphase HeLa cells. Traffic 5, 595-605.
- Tarrago-Trani, M. T., and B. Storrie. 2004. A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose-agarose resin. Prot. Exp. Pur. 38, 170-176.
- Jiang, S., and B. Storrie. 2005. Cisternal rab proteins regulate Golgi apparatus redistribution in response to hypotonic stress. Mol. Biol. Cell. (In press).
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Progress 10/01/02 to 09/30/03
Outputs
Our goal is to characterize organelle assembly within the secretory pathway. The pathway is the route by which milk is secreted from cells and plant proteins are directed to storage vacuoles in seeds. Research focuses on the Golgi apparatus, the central organelle within this pathway. We find that Golgi apparatus resident proteins cycle and because of this that the Golgi apparatus is dynamically unstable. Our work last year established that all Golgi compartments are a dynamic derivative of the endoplasmic reticulum. 1) Using a GDP-restricted mutant of Sar1p, a small GTPase required for protein transport from the endoplasmic reticulum to the Golgi apparatus, we found that even so called Golgi matrix components were unstable in their distribution. 2) we used three different approaches to determine whether assembly of the Golgi apparatus from the endoplasmic reticulum is a sequential process in which potential nucleation requires functional ER-exit sites. The overall
outcome of these experiments provide evidence that the assembly of the Golgi apparatus can be separated into stages in which the formation of early (p27 and GM130) positive structures can be dissociated from that of subsequent cisternal enzyme positive structures. 3) To test if rab proteins act independently to promote Golgi apparatus to ER trafficking, we asked if there was crosstalk between rab33b and rab6, a trans Golgi rab that exists in at least two isoforms, rab6a and rab6a1. Titration of GDP-restricted rab33b, rab6a, or rab6a1 against low levels of the respective GTP-restricted rab inhibited substantially Golgi redistribution to the ER. GDP-restricted rab33b had no effect on GTP-restricted rab6a/a1 induced redistribution and correspondingly GDP-restricted rab6a/a1 had no effect on rab33b induced redistribution indicating that rab33b and rab6 act independently and presumably locally. Consistent with local action, cross-titration of GDP-restricted rab6a/a1 against the opposite
GTP-restricted isoform inhibited strongly the redistribution. Overexpression of GTP-restricted rab33b induced the redistribution of Golgi proteins located trans and medial Golgi while causing deformation of cis Golgi structure, indicating a more global effect. Co-expression of GDP-restricted rab33b or rab6a/a1 individually with mutant Sar1p failed to inhibit mutant Sar1p induced redistribution of Golgi proteins at levels sufficient to inhibit the effect of the respective GTP-restricted rab. When the GDP-restricted forms of the rab proteins were co-expressed as a cocktail with mutant Sar1p there was a partial, synergistic inhibition of Golgi protein redistribution consistent with the involvement of multiple rabs. BFA-induced Golgi disassembly is not fully observed in GDP-Rab33b or GDP-Rab6a injected cells. Juxtanuclear Golgi staining was particularly apparent in most injected cells with mixed-rab-GDP. We conclude Rab33b acts independently of rab6, i.e., locally within the Golgi
apparatus, and suggest that it may produce more global effects with respect to Golgi organization. Synergistic interactions of rab proteins indicate multiple parallel pathways for Golgi protein cycling.
Impacts
Discovery from this research has altered our understanding of how cells assemble organelles involved in secreting or moving materials out of the cell. This assembly process is called retrograde cycling within the secretory pathway provides a novel pathway for drug delivery to the cell interior.
Publications
- Stroud, W. J., S. Jiang, G. Jack, and B. Storrie. 2003. Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase. Traffic 4, 631-641.
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Progress 10/01/01 to 09/30/02
Outputs
We have investigated the relative response of Golgi 'matrix' proteins and glycosyltransferases to a GDP-restricted endoplasmic reticulum (ER) exit block. Matrix proteins include peripheral membrane proteins of the golgin family that complex directly or indirectly with integral Golgi membrane proteins such as giantin. They may act as nucleators or exoskeletons for the assembly of the Golgi apparatus. HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block ER exit and then scored for the distribution of GM130, a cis located golgin; p27, a member of the p24 family of proteins; giantin; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2). Following a 6 h expression period, redistribution of all Golgi associated proteins exhibited a similar dependence on plasmid concentration. All proteins lost their compact, juxtanuclear distribution and displayed characteristics of ER/cytoplasmic accumulation.
There were no differences in the redistribution kinetics of GM130 and GalNAcT2. Expression of Sar1pT39N displaced the COPII coat protein Sec13p from ER exit sites without disturbing the overall distribution of ER membrane. The medial golgin, GRASP55, accumulated together with Sec13p in what may well be cytoplasmic inclusion bodies. Tagged Sar1pT39N accumulated in a uniform staining pattern in association with ER membranes. These observations suggest a Sar1pT39N induced disruption of all COPII rich ER exit sites. We conclude that Golgi matrix proteins are as unstable in their juxtanuclear distribution as Golgi glycosyltransferases and suggest that self-organization of Golgi membrane proteins and lipids may drive assembly of a dynamic Golgi apparatus.
Impacts
This research strongly suggests that the central organelle within the secretory pathway of cells is a derivative of the ER rather than a stable, lowest energy organelle. This work helps to change the concept of what we think of as an organelle. In addition, this research forms the scientific basis of a related research project to use retrograde trafficking of protein toxin subunits as a means to kill undesired cells in humans and animals.
Publications
- Miles, S., McManus, H., Forsten, K. E., and Storrie, B. 2001. Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block. J. Cell Biol. 155:543-555.
- Valsdottir, R., Hashimoto, H., Ashman, K., Koda, T., Storrie, B. and Nilsson. T. 2001. Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER. FEBS Lett, 508:201-209.
- Storrie, B. and Nilsson, T. 2002. The Golgi apparatus: balancing new with old. Traffic 3:521-529.
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Progress 10/01/00 to 09/30/01
Outputs
We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi proteins to an ER exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor or by plasmid encoded expression of the same dominant negative Sar1p. All Golgi proteins examined lost juxtanuclear Golgi-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI and p115. Giantin, GM130 and GalNAcT2 relocated with approximately equal kinetics. Distinct ER
accumulation could be demonstrated for all integral membrane proteins. ER accumulated Golgi proteins were functional. The role of rab33b, a Golgi specific regulatory protein, was investigated. Microinjection of rab33b mutants stabilized in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and a destabilization of Golgi apparatus structure, Golgi glycosyltransferases recycled to the ER. We conclude that the entire Golgi apparatus is a dynamic structure regulated by rab proteins such as rab33b. We suggest that most, if not all, Golgi integral membrane proteins cycle through ER in interphase cells.
Impacts
This research strongly indicates that the central organelle within the secretory pathway is a derivative of the ER rather than a stable, locked-in-place organelle. It changes the concept of what we think of as an organelle. In addition, this research forms the scientific basis of a related research project to use retrograde trafficking of protein toxin fragments as a means to kill undesired cells in humans and animals.
Publications
- Miles, S., McManus, H., Forsten, K.E. and Storrie, B. 2001. Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block. J. Cell Biol., in press.
- Valsdottir, R., Hashimoto, H., Ashman, K., Koda, T., Storrie, B. and Nilsson, T. 2001. Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER. FEBS Lett., in press.
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Progress 10/01/99 to 09/30/00
Outputs
Experiments were focussed on testing whether retrograde trafficking of enzymes between the Golgi apparatus and endoplasmic reticulum (ER) is a normal, physiological process or not. We also asked whether or not such trafficking is typical of all Golgi proteins. We found in wild type, cultured human cells (HeLa) that the endogenous Golgi proteins, giantin, GalNAcT2, GalT, and TGN46, accumulated in the ER through retrograde trafficking when ER exit is blocked by a Sar1p mutant. We found, using HeLa cells overexpressing a wide range of Golgi proteins, that the same ER accumulation is observed for all proteins including both integral and peripheral Golgi membrane proteins. We concluded that the entire Golgi apparatus cycles through the ER. We observed in photobleaching experiments that Golgi proteins cycle to the ER in the absence of any Sar1p mutation. Therefore, we concluded that Golgi protein cycling through the ER is a normal, physiological process.
Impacts
Proteins, such as toxins, enter cells and are altered in specific ways. Hence, toxicity and cell death are related to processing of the toxic protein. This research forms the scientific basis to explore retrograde trafficking of protein toxin fragments as a basis for killing undesired cells in humans and animals.
Publications
- Storrie, B., Pepperkok, R. and Nilsson, T. 2000. Breaking the COPI monolopy on Golgi recycling. Trends Cell Biol. 10, 385-391.
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