SGTA regulates the cytosolic quality control of hydrophobic substrates.
Bottom Line: Our data show that the BAG6 complex and SGTA compete for cytosolic MLPs by recognition of their exposed hydrophobicity, and the data suggest that SGTA acts to maintain these substrates in a non-ubiquitylated state.Hence, SGTA might counter the actions of BAG6 to delay the ubiquitylation of specific precursors and thereby increase their opportunity for successful post-translational delivery to the endoplasmic reticulum.However, when SGTA is overexpressed, the normally efficient removal of aberrant MLPs is delayed, increasing their steady-state level and promoting aggregation.
Affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.Show MeSH
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Mentions: Our previous work suggested that SGTA influences the degradation of MLPs by antagonising the actions of the BAG6 complex (Leznicki and High, 2012), and we therefore next asked whether OPG–TASK85 is a substrate for BAG6-mediated quality control. An siRNA-mediated knockdown of BAG6 strongly enhances the steady-state level of OPG–TASK85 (Fig. 3A, cf. lanes 4 and 5) but, as with SGTA coexpression, there is little effect upon the R4 variant that lacks a functional hydrophobic degron (Fig. 3A, cf. lanes 6 and 7). The actions of the BAG6 complex rely on its ability to promote the polyubiquitylation of its substrates (Hessa et al., 2011; Minami et al., 2010); hence, replacing the lysine residues present in OPG–TASK85 both enhances steady-state levels of the resulting OPG–TASK85 ΔK in comparison to the parental OPG−TASK85 and negates the effect of a BAG6 knockdown (Fig. 3A, cf. lanes 4, 5, 8 and 9). We conclude that OPG−TASK85 is normally degraded through a pathway that requires the recognition of a contiguous stretch of hydrophobicity, is facilitated by the BAG6 complex and involves the ubiquitylation of its lysine side chains. A direct role for the BAG6 subunit of the BAG6 complex in triaging OPG–TASK85 was underlined by a physical interaction between these two components that was not observed with OPG–TASK85 R4 (see Fig. 3B,C, cf. lanes 5 to 8). Furthermore, when the BAG6 subunit is exogenously expressed in HeLaM cells, a substantial proportion of the protein is found in the nucleus (Manchen and Hubberstey, 2001), resulting in a parallel redistribution of OPG–TASK85 from the cytosol to the nucleus of cells that coexpress both components (Fig. 3Di). Nuclear relocalisation of OPG–TASK85 is not observed with a BAG6 mutant bearing a non-functional nuclear localisation signal (Fig. 3Dii) or with the OPG–TASK85 R4 variant (Fig. 3Diii).
Affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.