There are two long-term objectives in this proposal. One is to better comprehend how certain bacterial and plant protein toxins exploit pathways of membrane traffic to enter the cytosol of eukaryotic cells. Information about the entry process is important to understand how pathogenic toxins work and also may provide insight into basic cellular processes. The second objective is to capitalize on the properties of protein toxins to develop applications of therapeutic value. There are four specific aims:
AIM 1, analysis of toxin entry into CHO cell mutants of the ldlF group. ldlF mutants express a temperature-sensitive defect in epsilon-COP, a subunit of non-clathrin coatomers. These cells serve as a model system to investigate whether toxin access to the cytosol requires epsilon-COP.
AIM 2, the interaction of precleaved exotoxin A (ETA) with cells. ETA must be proteolytically cleaved to enter the cytosol. We are studying here the consequences of precleaving the toxin before adding it to cells.
AIM 3, the effect of covalently attaching apyrase to ETA. The objective of this aim is to determine whether ETA enters the endoplasmic reticulum before entering the cytosol. If it does, then ETA should carry attached apyrase to the endoplasmic reticulum, which should have detectable consequences.
AIM 4, studies of a variant of ETA that has a free sulfhydryl near the carboxyl terminus. ETA with a cysteine residue inserted near the carboxyl terminus has much-reduced cytotoxicity. The objective of this aim is to understand why.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034297-12
Application #
2684787
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Haft, Carol Renfrew
Project Start
1985-01-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas-Dallas
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Richardson
State
TX
Country
United States
Zip Code
75080
Chen, Alice; AbuJarour, Ramzey J; Draper, Rockford K (2003) Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI. J Cell Sci 116:3503-10
Chen, Alice; Hu, Tonghuan; Mikoryak, Carole et al. (2002) Retrograde transport of protein toxins under conditions of COPI dysfunction. Biochim Biophys Acta 1589:124-39
Draper, R K; Hudson, R T; Hu, T (2001) Use of aminoglycoside antibiotics and related compounds to study ADP-ribosylation factor (ARF)/coatomer function in Golgi traffic. Methods Enzymol 329:372-9
Hu, T; Kao, C Y; Hudson, R T et al. (1999) Inhibition of secretion by 1,3-Cyclohexanebis(methylamine), a dibasic compound that interferes with coatomer function. Mol Biol Cell 10:921-33
Hudson, R T; Draper, R K (1997) Interaction of coatomer with aminoglycoside antibiotics: evidence that coatomer has at least two dilysine binding sites. Mol Biol Cell 8:1901-10
Corboy, M J; Draper, R K (1997) Elevation of vacuolar pH inhibits the cytotoxic activity of furin-cleaved exotoxin A. Infect Immun 65:2240-2
Kao, C Y; Draper, R K (1992) Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells. J Cell Biol 117:701-15
Wang, R H; Colbaugh, P A; Kuo, P et al. (1992) Novel method for isolating mammalian cells defective in fluid-phase endocytosis. Somat Cell Mol Genet 18:543-51
Park, J E; Draper, R K; Brown, W J (1991) Biosynthesis of lysosomal enzymes in cells of the End3 complementation group conditionally defective in endosomal acidification. Somat Cell Mol Genet 17:137-50
Draper, R K; Goda, Y; Brodsky, F M et al. (1990) Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro. Science 248:1539-41

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