Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells

Abstract

The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-β-cyclodextrin (MβCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MβCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.

Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

“…Such partial association has been observed in several other systems and with several other proteins (13,31,33). It may reflect that the association with lipid rafts is only transient or is specifically regulated.…”

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Likewise, a portion of Ntcp protein localizes to membrane rafts in mouse liver (Molina et al, 2008). Within the mouse intestine, Pept1 protein colocalizes with markers of membrane rafts (Nguyen et al, 2007). Using rat liver, it has been demonstrated that the localization of ABC family transporters (Abcg5, Bsep, Mrp2, Mdr2) is predominantly in "lubrol-microdomains" that contain the majority of canalicular cholesterol and phospholipids, as well as caveolin-1 and dipeptidyl peptidase IV proteins (Ismair et al, 2009).…”

“…It should be noted that depletion of cholesterol yields conflicting results on Pept1 activity in polarized intestinal epithelial cells (increased activity) compared with intestinal apical membrane vesicles (decreased activity) (Nguyen et al, 2007). As future studies of transporters and lipid rafts are designed, these findings should be taken into consideration.…”

The Human Concentrative Nucleoside Transporter-3 C602R Variant Shows Impaired Sorting to Lipid Rafts and Altered Specificity for Nucleoside-Derived Drugs

“…Associations between lipid-raft localization and functional activity have been described for other transporter proteins. In particular, it has been shown that cholesterol depletion of nonpolarized cells results in a decrease in NHE3 and hPept1 activities (Murtazina et al, 2006;Nguyen et al, 2007). Our analysis showed that lipid-raft localization of the hCNT3C602R variant was impaired.…”

“…Amino acid changes in transmembrane domains can alter the affinity of transporter proteins for lipid rafts, which in turn may affect their biological activity (Murtazina et al, 2006;Nguyen et al, 2007). To first establish the distribution of hCNT3 in membrane lipid domains, for which no information is currently available, we assessed hCNT3 protein levels in purified lipid microdomains by Western blot analysis.…”