Transcytosis of Aminopeptidase N in Caco-2 Cells Is Mediated by a Non-cytoplasmic Signal

doi:10.1074/jbc.270.39.22933

Transcytosis of Aminopeptidase N in Caco-2 Cells Is Mediated by a Non-cytoplasmic Signal (*)

From the Department of Medical Biochemistry and Genetics, Biochemistry Laboratory C, University of Copenhagen, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

^§ To whom correspondence should be addressed. Tel.: 45-35327789; Fax: 45-35367980.

Abstract

In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P.(1990) Cell 60, 429-437). The aim of this study was to determine the importance of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N was observed. This indicates that transcytosis of aminopeptidase N from the basolateral to the apical membrane does not occur by default transport but involves an active sorting mechanism.

Footnotes

↵* This work was supported by grants from the Danish Medical Research Council, The Danish Cancer Society, and the Lundbeck Foundation. This project was a part of a program under the Biomembrane Research Center, Aarhus University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MDCK

Madin-Darby canine kidney

BSA

bovine serum albumin

MESNA

2-mercaptoethanesulfonic acid

PBS

phosphate-buffered saline

s-NHS-biotin

sulfo-N-hydroxysuccinimido-biotin

s-NHS-SS-biotin

sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate

PAGE

polyacrylamide gel electrophoresis

APN

aminopeptidase N.
↵²L. K. Vogel, H. Sjöström, and O. Norén, unpublished observation.
- Received May 11, 1995.