Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins

Abstract

TPR proteins modulate the activity of molecular chaperones. Here, we describe the S. cerevisiae TPR protein Sgt2 as interaction partner of Ssa1 and Hsp104 and as a component of the GET pathway by interacting with Get5. The GET pathway mediates the sorting of tail-anchored (TA) proteins, harboring a C-terminal trans-membrane segment, to the ER membrane. S. cerevisiae sgt2Δ cells show partial defects in TA protein sorting. Sgt2 activity in vivo relies on its N- and C-terminal domains, whereas the central TPR domain and thus chaperone interactions are dispensable. We show that TA protein sorting defects are more severe in sgt2Δ get5Δ mutants compared to single knockouts. Furthermore, overproduction of Sgt2 becomes toxic to get3Δ but not to get5Δ cells. Together, these findings indicate an additional, Get5-independent role of Sgt2 in TA protein sorting, pointing to parallel pathways of substrate delivery to Get3.

Structural and Functional Characterization of Ybr137wp Implicates Its Involvement in the Targeting of Tail-Anchored Proteins to Membranes

“…Interestingly, the severity of the viability defect for each strain was dependent on the missing protein, as follows: Get3 Ͼ Get5 Ͼ Sgt2. This order was also observed for GFP-Sed5 mislocalization and hygromycin B sensitivity in GET protein-deleted cell lines (17).…”

“…Previous studies indicated that the acidic C-terminal DDLD motif of Hsp104 is responsible for its association with other proteins (17). We noted that Ybr137wp also contains an acidic motif, EEDL, in its C-terminal region.…”

“…Coimmunoprecipitation studies have shown that Sgt2 associates with the Get3, Get4, and Get5 proteins and chaperone proteins such as Hsp104 and Hsp70 (20). Interestingly, a then-novel protein encoded by ybr137w was also found to associate with the Sgt2 complex (13,17,20). To characterize the interaction between Ybr137wp and Sgt2, we performed an in vitro binding assay using purified recombined proteins.…”

“…Its yeast homolog, Get3, and related proteins (Get1, Get2, Get4, Get5, and Sgt2) in the GET system were subsequently identified (9,11,13,20,51). Substantial evidence has demonstrated that Sgt2 acts upstream of the GET pathway (13,17). Sgt2 is complexed with Get4 and Get5.…”

“…6). Given the facts that removal of the TPR domain on Sgt2 has no effect on the distribution of Sed5 (a Golgi membraneresiding TA protein) in yeast (17) and that Sgt2 with a mutation at the TPR domain can still load the TA protein Sec22 onto Get3 in vitro (13), the biological function of the TPR domain of Sgt2 and the role of its association with Ybr137wp in the GET pathway remain elusive. Indeed, the role of Ybr137wp in the delivery of TA proteins was assessed by using the temperature-sensitive phenotype commonly found in yeast when any protein in its GET pathway is deleted (Fig.…”