Investigating the role of TRC40 in post-translational protein delivery and quality control

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.