The long-term goal of this project is to elucidate the mechanism of the Guided Entry of Tail-anchored proteins (GET) pathway. This pathway targets tail-anchored (TA) proteins destined for the secretory pathway for post-translational insertion into the endoplasmic reticulum (ER) membrane. The central component of this pathway is the chaperone Get3. We know that once Get3-TA protein complexes are formed they are recruited to the ER membrane for insertion. Beyond this conceptual framework, we still know very little about how Get3 finds its TA protein substrates and by what mechanism they are inserted into the ER membrane. To gain a deeper mechanistic understanding of the GET pathway, this project will determine (1) how an upstream chaperone complex that we identified delivers TA proteins with ER-targeting signals to Get3, and (2) how TA proteins associated with Get3 are inserted by two membrane components of the pathway that we have reconstituted into proteoliposomes. These studies will be performed in budding yeast because this model organism has facile genetics, is biochemically tractable, and cell biologically accessible to imaging using fluorescence microscopy. The proposed studies will reveal the mechanism of a conserved targeting pathway that enables the biogenesis of hundreds of TA proteins in human cells. From an academic standpoint, the GET pathway is interesting because it accurately distinguishes the membrane targeting signals of TA proteins destined for the secretory pathway form those of mitochondrial TA proteins. Furthermore, this pathway uses a novel membrane insertion machinery, distinct from the canonical Sec61 protein translocation channel, to integrate transmembrane domains into the ER lipid bilayer. From a practical standpoint, these studies will shed light on how tail-anchored Bcl2 proteins regulate apoptosis by partitioning between the ER and the mitochondrial outer membrane. Lastly, Asna1, the metazoan Get3 homolog, has emerged as a critical factor for cellular sensitivity to cisplatin. Thus, the proposed studies will inform pharmacological approaches for manipulating Asna1 activity to minimize drug resistance and enhance existing cisplatin chemotherapies.
This project addresses how cells use a novel """"""""molecular machine"""""""" to sort hundreds of proteins to their appropriate intracellular locations. Abnormal regulation of one of the machine components makes cells more sensitive to cisplatin, a common cancer chemotherapeutic. Thus, manipulation of this component using drugs will help reduce resistance to cisplatin during many chemotherapies.
|Weir, Nicholas R; Kamber, Roarke A; Martenson, James S et al. (2017) The AAA protein Msp1 mediates clearance of excess tail-anchored proteins from the peroxisomal membrane. Elife 6:|
|Zalisko, Benjamin E; Chan, Charlene; Denic, Vladimir et al. (2017) Tail-Anchored Protein Insertion by a Single Get1/2 Heterodimer. Cell Rep 20:2287-2293|
|Wang, Fei; Chan, Charlene; Weir, Nicholas R et al. (2014) The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase. Nature 512:441-4|
|Denic, Vladimir; Dötsch, Volker; Sinning, Irmgard (2013) Endoplasmic reticulum targeting and insertion of tail-anchored membrane proteins by the GET pathway. Cold Spring Harb Perspect Biol 5:a013334|
|Denic, Vladimir (2012) A portrait of the GET pathway as a surprisingly complicated young man. Trends Biochem Sci 37:411-7|
|Wang, Fei; Whynot, Andrew; Tung, Matthew et al. (2011) The mechanism of tail-anchored protein insertion into the ER membrane. Mol Cell 43:738-50|
|Denic, Vladimir (2011) No country for old misfolded glycoproteins. Mol Cell 42:715-7|
|Stefer, Susanne; Reitz, Simon; Wang, Fei et al. (2011) Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex. Science 333:758-62|
|Wang, Fei; Brown, Emily C; Mak, Gary et al. (2010) A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum. Mol Cell 40:159-71|