We have developed a new model system to access directly intracellular organelles of the eucaryotic cell. The plasma membrane of the eucaryotic cell can be selectively perforated to produce semi-intact cells, a cell population in which organelles of the secretory pathway (nucleus, endoplasmic reticulum (ER), Golgi, and plasma membrane) are retained in an intact, functional form and directly accessible to a wide range reagents and macromolecules. It is the broad objective of the proposal to use this model system to study in vitro the transport of protein between the ER and the cis Golgi compartment. Transport of protein from the ER to the Golgi occurs by the budding and fusion of carrier vesicles. Vesicle formation requires ATP and as yet unspecified soluble and membrane-associated components. Fusion of carrier vesicles to the Golgi compartment occurs through a series of intermediate transport steps which require GTP hydrolysis and Ca2+. We propose to specifically examine the role of the rab gene family of synthesis of peptide reagents encoding functional domains of the rab gene family. Peptides will be used as chemical analogs to inhibit transport in order to elucidate rab protein function and to prepare domain specific polyclonal and monoclonal antibodies to inhibit ER to Golgi transport in vitro. These antibodies will be used as reagents to study the role of low-molecular weight GTP binding proteins in ER to Golgi transport and to identify potentially new members of this family. In addition, recombinant rab protein will be prepared using both procaryotic and eucaryotic expression vectors for analysis of the role of known rab gene products in transport in vivo and in vitro. Site-directed binding, hydrolysis and membrane-association in transport. We will also explore the role of a newly discovered Ca2+-dependent step(s) in vesicular transport which is likely to require novel Ca2+-binding protein(s) for vesicle fusion to the Golgi compartment. These studies will include purification and characterization of 3 sequential vesicular intermediates which accumulate transported protein under 3 conditions: (1) reduced temperature (15degreesC), (2) in the absence of GTP hydrolysis, and (3) in the absence of free Ca2+. Many medically important diseases result from defects in transport between intracellular organelles and the cell surface. These include lysosomal storage diseases, familial hypercholesterolemia, cystic fibrosis, and cancer. Establishing a model in vitro system for the study of transport events occurring along the secretory pathway will have a major impact on our understanding of the biochemistry of these diseases as a result of understanding the basic mechanisms fundamental to trafficking of protein between organelles of the eucaryotic cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042336-05
Application #
2181303
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Amaral, Margarida D; Balch, William E (2015) Hallmarks of therapeutic management of the cystic fibrosis functional landscape. J Cyst Fibros 14:687-99
Roth, Daniela Martino; Hutt, Darren M; Tong, Jiansong et al. (2014) Modulation of the maladaptive stress response to manage diseases of protein folding. PLoS Biol 12:e1001998
Hutt, Darren M; Balch, William E (2013) Expanding proteostasis by membrane trafficking networks. Cold Spring Harb Perspect Med 3:1-21
Powers, Evan T; Balch, William E (2013) Diversity in the origins of proteostasis networks--a driver for protein function in evolution. Nat Rev Mol Cell Biol 14:237-48
Pottekat, Anita; Becker, Scott; Spencer, Kathryn R et al. (2013) Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release. Cell Rep 4:921-30
Hutt, Darren M; Balch, William E (2013) Expanding proteostasis by membrane trafficking networks. Cold Spring Harb Perspect Biol 5:
Hutt, Darren M; Roth, Daniela Martino; Chalfant, Monica A et al. (2012) FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability. J Biol Chem 287:21914-25
Bouchecareilh, M; Balch, W E (2012) Proteostasis, an emerging therapeutic paradigm for managing inflammatory airway stress disease. Curr Mol Med 12:815-26
Bouchecareilh, Marion; Hutt, Darren M; Szajner, Patricia et al. (2012) Histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA)-mediated correction of ýý1-antitrypsin deficiency. J Biol Chem 287:38265-78
Coppinger, Judith A; Hutt, Darren M; Razvi, Abbas et al. (2012) A chaperone trap contributes to the onset of cystic fibrosis. PLoS One 7:e37682

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