Abstract
ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.
Highlights
Ing and organelle structure [1, 2]
Additional data suggest that ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is required for cell surface localization of E-cadherin because in the absence of ARFRP1, E-cadherin is dislocated from the plasma membrane, and cell adhesion is markedly reduced in vivo and in vitro
Distribution of E-cadherin Is Altered in Arfrp1Ϫ/Ϫ Embryos—We have previously shown that deletion of Arfrp1 in mice results in embryonic lethality because of the failure of differentiating epiblast cells to form the mesoderm
Summary
Antisera—The polyclonal antibody against recombinant GST-ARFRP1 was described before [3] and used for immunohistochemistry [10]. Monoclonal antibody against E-cadherin (clone HECD-1) was used in a dilution of 1:400 for immunofluorescence analysis (Zymed Laboratories Inc.). Cells from each of the surviving clones were split and maintained in the presence or absence of doxycycline. After extensive washing with PBS, 0.1% (v/v) Tween 20, cells were incubated with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibodies in antibody diluent at room temperature for 30 min. After washing with PBS, 0.1% (v/v) Tween 20, cells were mounted in fluorescent mounting medium (Dako) and analyzed with a Leica TCS SP2 Laser Scan inverted microscope.
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