Recently, we discovered a cytosolic TMD recognition complex (TRC) that selectively interacts with TA proteins destined for the ER membrane. A central component of TRC was identified as a highly conserved 40 kD ATPase and represents the first molecular factor in this widely used membrane protein insertion pathway. Other components that collaborate with TRC40 to mediate selective recognition, targeting, and insertion of TA proteins into the ER are unknown. We are now taking various approaches to identify these additional factor(s) and reconstitute the targeting reaction for TA proteins with defined components. Achieving these goals will define the core machinery and functions for a fundamental protein trafficking pathway and pave the way for future mechanistic and structural analyses. In the past year, we have made substantial progress towards these goals on two fronts. First, in collaboration with Robert Keenan, the high resolution crystal structure of TRC40 homologs were solved in multiple conformations. When combined with biochemical analysis, this work helped provide a mechanistic understanding how how TRC40 recognizes substrates and how it might function to bind and release them in an ATP-dependent manner. Second, we identified additional factors in this pathway. Most notably, we identified a conserved chaperone complex that seems to facilitate substrate capture by TRC40. This chaperone complex is novel and may have broader roles that are currently being studied. Our ongoing efforts are aimed toward identifying additional factors, as well as understanding the mechanism of function of the currently known factors.
|Mariappan, Malaiyalam; Mateja, Agnieszka; Dobosz, Malgorzata et al. (2011) The mechanism of membrane-associated steps in tail-anchored protein insertion. Nature 477:61-6|
|Shao, Sichen; Hegde, Ramanujan S (2011) A flip turn for membrane protein insertion. Cell 146:13-5|
|Sherrill, John; Mariappan, Malaiyalam; Dominik, Pawel et al. (2011) A conserved archaeal pathway for tail-anchored membrane protein insertion. Traffic 12:1119-23|
|Shao, Sichen; Hegde, Ramanujan S (2011) Membrane protein insertion at the endoplasmic reticulum. Annu Rev Cell Dev Biol 27:25-56|
|Sharma, Ajay; Mariappan, Malaiyalam; Appathurai, Suhila et al. (2010) In vitro dissection of protein translocation into the mammalian endoplasmic reticulum. Methods Mol Biol 619:339-63|
|Mariappan, Malaiyalam; Li, Xingzhe; Stefanovic, Sandra et al. (2010) A ribosome-associating factor chaperones tail-anchored membrane proteins. Nature 466:1120-4|
|Mateja, Agnieszka; Szlachcic, Anna; Downing, Maureen E et al. (2009) The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461:361-6|