Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting

Ashley J Evans*, James Daly, Anis N K Anuar, Boris Simonetti, Pete J Cullen *

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

29 Downloads (Pure)

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.
Original languageEnglish
Article numberjcs246033
JournalJournal of Cell Science
Volume133
DOIs
Publication statusPublished - 3 Aug 2020

Keywords

  • ESCPE-1
  • VPS35
  • Endosome
  • Retromer
  • Knocksideways
  • GLUT1
  • CI-MPR
  • SNX5

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