Characterization of the interaction of Get3 with the membrane bound receptors Get1 and Get2
Final Report Abstract
The insertion of membrane proteins into the correct membrane of a cell is an important cellular task. A special class of membrane proteins, called tail-anchored proteins, consist of an N-terminal soluble domain and a single C-terminal transmembrane helix. Insertion of these tail-anchored proteins into membranes requires a specialized machinery that includes several soluble and membrane-bound components. After synthesis at the ribosome the transmembrane helix of the tail-anchored protein is loaded into a hydrophobic groove on the protein Get3. This protein interacts in an ATP-dependent manner with the two membrane bound receptors Get1 and Get2. While Get2 is important for recruiting Get3 to the membrane, the interaction with Get1 opens the closed conformation of Get3 and initiates the insertion process. In a previous study we have structurally characterized the interaction between the soluble domains of Get1 and Get2 with the TA-binding protein Get3. In this study we aimed at further investigating these interactions by solving the crystal structure of either the isolated membrane components or the complex of Get1/Get2 with Get3. We have created several expression constructs in Saccharomyces cerevisiae and have shown that all constructs are correctly targeted to the internal ER and Golgi membranes. We have established expression and purification protocols for several constructs, including fusion constructs of Get1 and Get2. Size exclusion chromatography has shown that all constructs behave well. Unfortunately, crystallization trials have resulted in small crystals which however do not diffract well so far. In parallel we have started to use electron microscopy to obtain structural information. First attempts using negative stain have been successful.
Publications
- (2014). ER Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET pathway. Cold Spring Harb Perspect Biol. 5(8)
Denic, V., Dötsch., V., and Sinning, I
(See online at https://doi.org/10.1101/cshperspect.a013334)
