Biomedical Engineering Reference
In-Depth Information
Chapter 11
Analysis of Conventional and Unconventional
Traffi cking of CFTR and Other Membrane Proteins
Heon Yung Gee , Joo Young Kim , and Min Goo Lee
Abstract
The cystic fi brosis transmembrane conductance regulator (CFTR) is a polytopic transmembrane protein
that functions as a cAMP-activated anion channel at the apical membrane of epithelial cells. Mutations in
CFTR
cause cystic fi brosis and are also associated with monosymptomatic diseases in the lung, pancreas,
intestines, and vas deferens. Many disease-causing CFTR mutations, including the deletion of a single
phenylalanine residue at position 508 (
ʔ
F508-CFTR), result in protein misfolding and traffi cking defects.
Therefore, intracellular traffi cking of wild-type and mutant CFTR has been studied extensively, and results
from these studies signifi cantly contribute to our general understanding of mechanisms involved in the
cell-surface traffi cking of membrane proteins. CFTR is a glycoprotein that undergoes complex
N-glycosylation as it passes through Golgi-mediated conventional exocytosis. Interestingly, results from
recent studies revealed that CFTR and other membrane proteins can reach the plasma membrane via an
unconventional alternative route that bypasses Golgi in specifi c cellular conditions. Here, we describe
methods that have been used to investigate the conventional and unconventional surface traffi cking of
CFTR. With appropriate modifi cations, the protocols described in this chapter can also be applied to stud-
ies investigating the intracellular traffi cking of other plasma membrane proteins.
Key words
Membrane protein, Unconventional traffi cking, Cystic fi brosis transmembrane conductance
regulator (CFTR), Surface biotinylation, Immunofl uorescence staining
1
Introduction
Defects in the folding of secretory proteins are often accompanied
by intracellular traffi cking defects, which underlie a number of
human diseases. A well-known example is the cystic fi brosis trans-
membrane conductance regulator (CFTR) (MIM 602421).
CFTR is a key transepithelial transport membrane protein in the
respiratory, gastrointestinal, and genitourinary systems. CFTR
functions as a cAMP-activated anion channel that permeates Cl
−
and HCO
3
−
and acts as a regulator of other membrane transport
proteins at the apical membrane of epithelial cells [
1
,
2
]. Loss of
CFTR function resulting from gene mutations causes a range of
epithelial disorders, including the classical form of cystic fi brosis (CF),
