Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes.
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 micro1B. 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 approximately 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.
Cancino
Jorge
J
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.
Torrealba
Carolina
C
Soza
Andrea
A
Yuseff
María Isabel
MI
Gravotta
Diego
D
Henklein
Peter
P
Rodriguez-Boulan
Enrique
E
González
Alfonso
A
D033961:Adaptor Protein Complex 1 / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011973:Receptors, LDL / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction; D013961:Thyroid Gland / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D000906:Antibodies; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011973:Receptors, LDL; D011990:Receptors, Transferrin; D014759:Viral Envelope Proteins; C089212:protein kinase D; D011493:Protein Kinase C