A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

Retrograde transport of membranes and proteins from the cell surface is essential to maintain homeostasis and compartment identity. Following internalization via clathrin-dependent or -independent endocytosis, lipid and protein cargoes first populate early endosomes from where they are further redirected either along the endo-lysosomal system, recycled to the plasma membrane, or targeted to the trans-Golgi network (TGN) compartment. A number of distinct sorting machineries have been implicated in retrograde transport from endosomes to the TGN, among them the AP-1/clathrin machinery. Apart from an involvement in retrograde transport, AP-1/clathrin carriers have a well-established function in cargo export from the TGN. Even though the concept of bidirectional traffic at the TGN-to-endosome interface is commonly accepted, there is still uncertainty about the precise contribution of AP-1 to retrograde transport, since the conclusions of most studies were based on altered receptor steady-state distribution or mislocalization analysis upon knockdown or knockout of AP-1. Their readouts may be misleading, because the observed phenotype may be an indirect consequence of long-term AP-1 depletion, the result of upregulation of alternative pathways to compensate for the reduced or missing protein, thereby potentially masking the true AP-1 phenotype.
To elucidate the involvement of AP-1 in endosome-to-TGN traffic, we set up a more generic approach allowing us to follow cargo molecules during their retrograde transport from the plasma membrane. To this end, we established a versatile nanobody-based approach conferring recombinant protein cargo to be tracked from the cell surface biochemically, by live cell imaging, and by electron microscopy. We engineered and bacterially expressed functionalized anti-GFP nanobodies fused to a sulfation consensus motif, to fluorophores, or to a peroxidase reporter. These functionalized nanobodies are specifically captured by EGFP-modified receptor proteins at the cell surface and transported piggyback to the receptor’s homing compartments. Using the sulfatable nanobody, we could biochemically determine the kinetics of bonafide sorting receptors, the MPRs, from the cell surface to the TGN. In combination with the knocksideways approach to look at the immediate and direct consequences of AP-1 inactivation, we could also show the role of AP-1/clathrin carriers in retrograde transport of MPRs from endosomes to the TGN. At the same time, however, we also evidenced that an AP-1 knockdown and knockout produced conflicting results when compared to acute inactivation strategies.
Collectively, the present study describes a versatile nanobody-based approach to analyze retrograde transport of cargo proteins from the cell surface, and moreover provides insights into the role of the AP-1/clathrin machinery in retrograde transport.