Yes / Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation. / BBSRC [grant number: BB/L006510/1] and the Wellcome Trust [grant number: 092107/Z/10/Z]. K.K. was supported by the UPStream network [EU, FP7, ITN project 290257]

Membrane compartmentalisation allows eukaryotic cells to perform complex processes by combining dedicated sets of proteins in the same organelle. To achieve this, the cell must first target the appropriate proteins, primarily synthesised on cytosolic ribosomes, to the correct subcellular location. Components of the secretory pathway/endomembrane system begin this journey via their signal sequence-dependent delivery to the endoplasmic reticulum (ER). These ER targeting signals are hydrophobic, and typically function whilst the protein is being synthesised, via a so-called 'co-translational' pathway. However, some hydrophobic signals can also facilitate post-translational protein targeting to the ER, or initiate regulated protein degradation in the cytosol. Tail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain that functions as both their subcellular targeting signal and membrane anchor. Recent evidence suggests that the canonical TRC40 pathway, through which mammalian TA proteins are delivered to the ER, may not be essential in vivo. In this thesis, I provide functional evidence for the existence of an orthologous SRP-independent (SND) pathway in mammalian cells and identify roles for both the signal recognition particle (SRP)-mediated pathway and presumptive mammalian SND pathway in the biogenesis of TA proteins. I conclude that although TRC40 normally plays a role in TA protein biogenesis, it is not essential, and speculate that these alternative pathways make a significant contribution to the apparent redundancy of the TRC40 pathway in vivo. The soluble components that act upstream of TRC40 during protein biogenesis also play an important role in the recognition and selective degradation of hydrophobic membrane and secretory proteins that mislocalise to the cytosol. I now provide preliminary evidence that TRC40 appears to exhibit dual functionality, having a non-essential role in TA protein delivery, whilst also contributing to protein quality control by acting as a putative holdase. My data suggest that both TRC40 and BAG6 can influence the proteasomal degradation of a novel class of substrates, which I have termed the aberrant short secretory proteins.

Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1