Submitted on July 23, 2007
Accepted on October 8, 2007
Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and non-polarized cells
Hang Thi Thu Nguyen1, Laetitia Nadia Charrier-Hisamuddin2, Guillaume Dalmasso2, Abel Hiol3, Shanthi V Sitaraman4, and Didier Merlin5*
1 Medicine, Emory University, Atlanta, Georgia, United States
2 Medicine/ GI division, Emory University, Atlanta, Georgia, United States
3 Cell Biology, Paul-Cezanne, Marseille, Marseille, France
4 Division of Digestive Diseases, Dept. of Medicine, Emory University, Atlanta, Georgia, United States
5 Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia, United States
* To whom correspondence should be addressed. E-mail: dmerlin{at}emory.edu.
The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in non-polarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs), polarized and non-polarized cells, as well as the 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 non-polarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 co-localized with the lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-
-cyclodextrin (MCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and non-polarized 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 non-polarized cells.
