Retrieval and recycling of proteins in the endosomal sorting pathway are orchestrated by several complexes, such as retromer, a heterotrimer composed of VPS26, VPS35 and VPS29, and endosomal SNX-BAR sorting complex for promoting exit-1 (ESCPE-1). Although retromer acts in the sorting of numerous proteins, its role in the retrograde endosome-to-trans Golgi network (TGN) trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) is still controversial. Now, Peter Cullen, Ashley Evans and colleagues (Evans et al., 2020) developed a system to rapidly inactivate retromer and ESCPE-1 in order to clarify their contribution to protein sorting. By adapting the knocksideways technique, they quickly trapped and inactivated either the retromer subunit VPS35 or the ESCPE-1 subunit sortin nexin 5 (SNX5) on peroxisomes of HeLa and H4 human neuroglioma cells; this led to the fast inactivation of the respective complexes and thus allowed for a temporal resolution of the sorting process. Retromer inactivation led to a missorting phenotype of its known cargo GLUT1, which then accumulated at late endosomes and lysosomes, whereas that of CI-MPR remained unaltered. In contrast, inactivation of ESCPE-1 resulted in less accumulation of CI-MPR at the TGN and a redistribution away from the TGN to endosomes. This study thus not only defines ESCPE-1 as the main regulator of CI-MPR retrograde sorting, but also provides a new tool to study the kinetics of endosomal sorting components through their acute inactivation.
