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.
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 language | English |
|---|---|
| Article number | jcs246033 |
| Journal | Journal of Cell Science |
| Volume | 133 |
| DOIs | |
| Publication status | Published - 3 Aug 2020 |
Keywords
- ESCPE-1
- VPS35
- Endosome
- Retromer
- Knocksideways
- GLUT1
- CI-MPR
- SNX5