Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator.
Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.
Gentzsch
Martina
M
Mayo Clinic College of Medicine, S.C. Johnson Medical Research Center, Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, Arizona 85259, USA.
Chang
Xiu-Bao
XB
Cui
Liying
L
Wu
Yufeng
Y
Ozols
Victor V
VV
Choudhury
Amit
A
Pagano
Richard E
RE
Riordan
John R
JR
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013696:Temperature; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
C505032:CFTR protein, human; D000939:Epitopes; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020691:rab GTP-Binding Proteins