Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin.

Glucose transporters are gatekeepers of cellular glucose metabolism. Understanding how their activity is regulated can provide insight into mechanisms of glucose homeostasis and diseases arising from dysregulation of glucose transport. Glucose stimulates endocytosis of the human glucose transporter GLUT1, but several important questions remain surrounding the intracellular trafficking itinerary of GLUT1. Here, we report that increased glucose availability triggers lysosomal trafficking of GLUT1 in HeLa cells, with a subpopulation of GLUT1 routed through ESCRT-associated late endosomes. This itinerary requires the arrestin-like protein TXNIP, which interacts with both clathrin and E3 ubiquitin ligases to promote GLUT1 lysosomal trafficking. We also find that glucose stimulates GLUT1 ubiquitylation, which promotes its lysosomal trafficking. Our results suggest that excess glucose first triggers TXNIP-mediated endocytosis of GLUT1 and, subsequently, ubiquitylation to promote lysosomal trafficking. Our findings underscore how complex coordination of multiple regulators is required for fine-tuning of GLUT1 stability at the cell surface.

Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin.

Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole.

Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase.

The Escherichia coli Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the E. coli QFR FrdA subunit. Using a ΔsdhE E. coli strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdAE245 residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdAE245Q have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdAE245 variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdAE245 residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.