1. Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting
- Author
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Anis N. K. Anuar, Boris Simonetti, James L. Daly, Peter J. Cullen, and Ashley J. Evans
- Subjects
Retromer ,Endosome ,Vesicular Transport Proteins ,Regulator ,Endosomes ,Biology ,03 medical and health sciences ,VPS35 ,ESCPE-1 ,0302 clinical medicine ,Humans ,Sorting Nexins ,VPS26A ,030304 developmental biology ,0303 health sciences ,Knocksideways ,Insulin-like growth factor 2 receptor ,Sorting ,Cell Biology ,Cell biology ,Protein Transport ,VPS29 ,CI-MPR ,SNX5 ,GLUT1 ,030217 neurology & neurosurgery ,Research Article ,HeLa Cells ,trans-Golgi Network - Abstract
Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex – a key regulator of retrograde CI-MPR sorting – revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting., Summary: Retromer, a master controller of endosomal cargo sorting, is deregulated in neurodegenerative disease. Here, we develop and apply a retromer knocksideways methodology to quantify endosomal cargo sorting upon acute perturbation.
- Published
- 2020