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

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.

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

“…Full-size  DOI: 10.7717/peerj.8751/ fig-5 While both the overlap with ERGIC53 and the GLUT4/DAPI ratio were decreased upon Sx6 knockdown, these did not reach significance (p~0.06 in both cases). This may reflect the relative capacity of the GOSR1/YKT6 pathway compared to the Sx6/Sx16 pathway in different cell types (Camus et al, 2020). These caveats notwithstanding, our data point to a role for the SNAREs GOSR1 and YKT6 in GLUT4 trafficking in human cells.…”

“…The relative balance of different endosomal pathways may differ between these cell types and thus reveal different facets of GLUT4 trafficking. Consistent with this, studies have suggested that GLUT4 trafficking differs between human and murine cells (Camus et al, 2020), and hence some distinctions between these two cell systems are not entirely unsurprising.…”

“…The HeLa cell line is an immortal cervical cancer cell line, originally isolated in 1951, and is the most widely investigated cell model (Macville et al, 1999). While HeLa cells do not contain any endogenous GLUT4, they do possess insulin sensitivity, and exhibit insulin-stimulated phosphorylation of Akt and AS160, two key signalling intermediates in insulin-stimulated GLUT4 translocation (Camus et al, 2020).…”

“…Recent studies have suggested that movement of GLUT4 from the ERGIC towards the GLUT4-storage compartment plays a key role in the trafficking of newly synthesised GLUT4 in human cells (Camus et al, 2020). The SNARE proteins BET1, BET1L, GOSR1, GOSR2, SEC22A, SEC22B, SEC22C, Stx5 and YKT6 are known to be involved in trafficking to or from the ERGIC (Zhang & Hong, 2001;Appenzeller-Herzog & Hauri, 2006;Adnan et al, 2019;Linders et al, 2019).…”

“…Recently, we and others have established HeLa cells expressing epitope-tagged GLUT4 as a model system which facilitate studies into aspects of GLUT4 biology by virtue of the ease of genetic manipulation of this commonly employed cell line (Kawase et al, 2006;Haga, Ishii & Suzuki, 2011;Kioumourtzoglou et al, 2015;Gulbranson et al, 2017;Camus et al, 2020). Here, we describe characterisation of these cells compared to 3T3-L1 adipocytes expressing the same GLUT4 reporter.…”