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Vol. 18, Issue 12, 4872-4884, December 2007

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Antibody to AP1B Adaptor Blocks Biosynthetic and Recycling Routes of Basolateral Proteins at Recycling Endosomes

Jorge Cancino^*,, Carolina Torrealba^*,, Andrea Soza^*,, María Isabel Yuseff^*,, Diego Gravotta, Peter Henklein, Enrique Rodriguez-Boulan, and Alfonso González^*,

*Departamento de Inmunología Clínica y Reumatología, Facultad de Medicina, and Centro de Regulación Celular y Patología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 6510260 Santiago, Chile; Millennium Institute for Fundamental and Applied Biology, 7780344 Santiago, Chile; Institute of Biochemistry Faculty of Medicine, Humboldt University, 10117 Berlin, Germany; and Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY 10021

Submitted June 13, 2007; Accepted September 11, 2007
Monitoring Editor: Keith Mostov

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit µ1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3–5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Alfonso González (agonzara{at}med.puc.cl).

Abbreviations used: AP, adaptor protein (complex); ARE, apical recycling endosomes; FRT, Fisher rat thyroid; LDLR, low-density lipoprotein receptor; RE, recycling endosomes; TfR, transferrin receptor; VSVG, vesicular stomatitis virus glycoprotein G.

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