Abstract
The processes controlling targeting of glucose transporters to apical and basolateral membranes of polarized cells are complex and not‐well understood. We have engineered SGLT1 and GLUT4 constructs linked to fluorescent proteins to highlight the differences in transporter expression and trafficking, in real time, in different cell types. Activity was assessed in parallel using a FRET glucose sensor. In COS cells and HEK cells, SGLT1 was distributed between the plasma membrane and intracellular compartments, but there was little expression in CHO cells. Trafficking was investigated using the lysosome inhibitors NH 4Cl (10 mmol/L) and chloroquine (150 μmol/L) and the proteasome inhibitors MG‐262 (1 μmol/L) and lactacystin (5 μmol/L). Lysosome inhibitors caused SGLT1 accumulation into intracellular bodies, whereas proteasome inhibitors induced SGLT1 accumulation in the plasma membrane, even in CHO cells. Our data suggest that a fraction of SGLT1 is rapidly degraded by lysosomes and never reached the plasma membrane; another fraction reaches the membrane and is subsequently degraded by lysosomes following internalization. The latter process is regulated by the ubiquitin/proteasome pathway, acting at a late stage of the lysosomal pathway. Using the cholesterol inhibitor Mβ CD (3 mmol/L), a dominant negative dynamin (K44A) and caveolin, we showed that SGLT1 internalization is lipid raft‐mediated, but caveolin‐independent. In contrast, GLUT4 internalization is dynamin‐dependent, but cholesterol‐independent. The physiological relevance of these data is discussed in terms of differential membrane compartmentalization of the transporters and expression under stress conditions.
Highlights
In intestinal absorptive cells and epithelial cells of the kidney, glucose is absorbed from the lumen, against its gradient, by the electrogenic Na/glucose cotransporter SGLT located in the brush-border or apical membrane – among the many isoforms, SGLT1 is most abundant in the small intestine, whereas SGLT2 is almost exclusively found in kidney (Wright et al 2011)
We generated SGLT1 fluorescent constructs to study the trafficking of SGLT1-Yellow Fluorescent Protein (YFP)/Cyan Fluorescent Protein (CFP) in real time in three different cell types
We observed that translocation of SGLT1-YFP/CFP is highly regulated and variable depending on the cell type
Summary
In intestinal absorptive cells and epithelial cells of the kidney, glucose (and galactose) is absorbed from the lumen, against its gradient, by the electrogenic Na/glucose cotransporter SGLT located in the brush-border or apical membrane – among the many isoforms, SGLT1 is most abundant in the small intestine, whereas SGLT2 is almost exclusively found in kidney (Wright et al 2011). Methylb-cyclodextrin (MbCD), which depletes cholesterol from membrane’s rich domains, is widely used to study membrane proteins that are internalized via caveolin-dependent or -independent pathways In this case, inhibition of lipid raft-dependent endocytosis by MbCD increases insertion of the selected proteins in the plasma membrane (Roepstorff et al 2002; Ivanov 2008). Ubiquitin tags membrane-bound receptors and channels such as EFGR, GHR, and connexin (Cx43) to facilitate their internalization and degradation by lysosomes In this instance, proteasome inhibitors may block a late stage of the lysosomal pathway and, as a result, increase protein recycling between the early endosome and the plasma membrane (Laing et al 1997; van Kerkhof et al 2001; Longva et al 2002; Alwan et al 2003). As with EGFR and GHR (van Kerkhof et al 2001; Longva et al 2002; Alwan et al 2003), lysosome inhibitors target SGLT1 to intracellular bodies and proteasome inhibitors increase membrane insertion of SGLT1, by inhibiting very likely the lysosomal pathway
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