It is proposed to study the nature of the interactions between membrane transport proteins and soluble proteins, making use of site-directed mutagenesis, biochemical techniques, and genetic suppression analysis. The system used as a model is a prokaryotic active transport system, the histidine periplasmic permease. This model system offers the following advantages: it has a complex structure (three membrane-bound proteins plus two soluble substrate-binding receptors), the genes for all five proteins have been cloned. and their sequences obtained, the proteins can be easily overproduced, the soluble proteins have been purified and crystallized, the membrane-bound proteins form a complex that can be solubilized and reconstituted into proteoliposomes, several crosslinking methods have been developed, and finally, but most importantly, genetic methods for the selection of suppressor mutations have been developed. The latter are a powerful tool that gives this particular system an advantage not usually available to studies in eukaryotic cells. Genetic suppression allows the selection and analysis of complementary mutations, thus yielding more readily a picture of two interacting protein surfaces. The proposed work will build an important base from which to proceed in the future to study interactions between the membrane-bound proteins, which are harder to analyze. This model system will be useful in establishing rules of conduct for these types of interaction that can be extrapolated to eukaryotic systems, such as protein hormone-receptor interactions especially, where a rapid genetic suppression analysis is not possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK043747-01
Application #
3245211
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1991-06-01
Project End
1994-05-31
Budget Start
1991-06-01
Budget End
1992-05-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704