CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis.

Stéphane M Camus,Christopher Esk,Nia J Bryant,Shaeri Mukherjee,Carmen Figueras-Novoa,Gaelle Boncompain,Franck Perez,Marine D Camus,L Amanda Sadacca,Gwyn W Gould,Anne Bigot,Frances M Brodsky,Dimitrios Kioumourtzoglou

doi:10.1083/jcb.201812135

Abstract

Glucose transporter 4 (GLUT4) is sequestered inside muscle and fat and then released by vesicle traffic to the cell surface in response to postprandial insulin for blood glucose clearance. Here, we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively secreted GLUT1 transporter and localize CHC22 to the ER-to-Golgi intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4, and sortilin, and downregulation of either p115 or CHC22, but not GM130 or sortilin, abrogates insulin-responsive GLUT4 release. This indicates that CHC22 traffic initiates human GLUT4 sequestration from the ERGIC and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.

Highlights

Summary: Blood glucose clearance relies on insulin-stimulated exocytosis of glucose transporter 4 (GLUT4) from sites of intracellular sequestration in muscle and fat
Endosomal sorting and retrograde transport through the trans-Golgi network is involved in this process, generating the GLUT4 storage compartment (GSC) (Shewan et al, 2003), which is a mixture of tubules and vesicles in which GLUT4 is sequestered in the absence of insulin
insulin treatment (Ins) human myocytes and adipocytes, GSC formation involves the non-canonical isoform of clathrin, CHC22, which is missing from rodents due to loss of the encoding gene (Wakeham et al, 2005)

Summary

Methods

Plasmids The HA-GLUT4-GFP construct was a gift from Dr Tim McGraw (Lampson et al, 2000). The plasmid encoding human GLUT1 was from OriGene. HA-GLUT4 and HA-GLUT1 were extracted using AcsI and EcoRI restriction enzymes and Cutsmart buffer from NEB and the agarose gel extraction kit from Qiagen. The inserts were ligated into the RUSH plasmid containing the ER Ii-hook fused to streptavidin, to generate the HA-GLUT4-SBP-GFP construct (Boncompain et al, 2012; Boncompain and Perez, 2013). In order to generate the HA-GLUT1-SBP-mCherry plasmid, we swapped the GFP tag in the HA-GLUT1-SBP-GFP for mCherry, using the SbfI and FseI restriction enzymes. The generation of plasmids encoding GFP-tagged CHC22 and CHC17 has been described elsewhere (Esk et al, 2010)

Results

Discussion

Conclusion