1. Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting.
- Author
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Evans, Ashley J., Daly, James L., Anuar, Anis N. K., Simonetti, Boris, and Cullen, Peter J.
- Subjects
- *
SURFACE defects , *MANNOSE , *ENDOSOMES - 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 CIMPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CIMPR 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. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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