Molecular basis of cargo sorting by endosomal trafficking machinery

Abstract

Faithful sorting of transmembrane proteins to their intended steady state localization is crucial for function of cellular organelles and homeostasis of an organism. Yet the vast majority of transmembrane proteins are synthesised in the endoplasmic reticulum (ER) despite the site of their function being within any number of a variety of other distinct organelles. Thus, within each cell there exists mechanism for the “un-mixing” of this otherwise gallimaufry of transmembrane proteins to their intended final destination. The early endosomal system serves as a checkpoint within the cell marking a critical junction between the Golgi apparatus, plasma membrane and endo-lysosomal pathway. It is at these early endosomal organelles that the sorting of a transmembrane protein determines if it is recycled for further use or degraded. Pathophysiological conditions from pathogens, rare genetic disorders and neurodegenerative conditions have all been causally linked to sorting and the endosomal system. Thus, understanding the mechanisms that drive sorting with molecular detail remains of vital importance not only to cellular function but also medicine. This study explores endosomal sorting at a molecular resolution using a combination of in vitro biochemistry and structural biology to understand the mechanisms of sorting of cargoes into and out of the endosomal system. The mechanism by which tethering of endosome-to-Golgi derived vesicles occurs remains incompletely understood. Here I characterize the binding of the tethering component, TBC1D23, directly to cargoes containing a novel acidic-TLY motif, suggesting the role of this tether in the sorting of carriers. While the sorting of acidic dileucine motifs is thought to be well understood, it remains unclear how different cargoes obtained different end point localisations despite containing similar motifs. Here I resolve the interaction of a dileucine motif bound to a fragment of the AP-2 complex at previously unobtained resolution, allowing visualization of not only the dileucine motif but also a previously unresolved water network mediating the reaction. This water network structurally explains the preferential endocytosis of a subset of cargo. Furthermore, the same water network is coordinated differently by the AP-3 complex to increase its affinity to the same subset of cargo, proposing a new mechanism of cargo sorting. Structural studies in understanding the AP-3 complex reveal a surprising lack of regulation in solution. Instead, I suggest the unfunctionalized δ-ear domain may serve to regulate the core through preventing recruitment to membrane by simultaneously breaking interaction sites for phosphoinositide lipids, Arf1 and dileucine cargo. Finally, I show that in vitro reconstitution of the AP-3 complex on synthetic membranes with PI35P2, Arf1 and cargo motifs is sufficient to drive assembly of highly ordered protein coated tubules, which may represent tubular carriers in vivo.