A Role for Acidic Residues in Di-leucine Motif-based Targeting to the Endocytic Pathway

doi:10.1074/jbc.270.34.19989

A Role for Acidic Residues in Di-leucine Motif-based Targeting to the Endocytic Pathway (*)

From the (1)R. W. Johnson Pharmaceutical Research Institute, San Diego, California 92121, the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, and the
(2) Department of Molecular Biology, the Scripps Research Institute, La Jolla, California 92037

^§Present address:
Dept. of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, U. K.
To whom correspondence should be addressed. Tel.: 44-1382-345097; Fax: 44-1382-201063; lpond{at}bad.dundee.ac.uk

↵^¶ Present address: Unité d'Immunité Cellulaire Antivirale, Institut Pasteur, 28 rue de Dr. Roux, Paris 75015, France.

Abstract

Recent reports have suggested that major histocompatibility complex class II molecules load peptide through a specialized compartment of the endocytic pathway and are targeted to this pathway by association with invariant chain (Iip31). Therefore we used a site-directed mutagenesis approach to determine whether Iip31 possesses novel protein targeting signals. Our results indicate that two di-leucine-like pairs mediate Iip31 targeting and that an acidic amino acid residue four or five residues N-terminal to each Iip31 di-leucine-like pair is required for endocytic targeting. Results from additional testing with hybrid Iip31 molecules indicate that the acidic residues N-terminal to di-leucine pairs are critical for accumulation of these molecules in large endocytic vesicles and in some cases provide a structure favorable for internalization. The acidic residues N-terminal to di-leucine pairs are important in some sequence contexts in providing a structure favorable for internalization, whereas in other contexts an acidic residue is critical for targeting to, and formation of, large endocytic vesicles. Although our results do not support the idea that Iip31 possesses unique protein targeting motifs, they do suggest that di-leucine motifs may be recognized as part of a larger secondary structure. In addition, our data imply that the targeting motif requirements for internalization may differ from the requirements for further transport in the endocytic pathway.

Footnotes

↵* This work was supported in part by National Institutes of Health (NIH) grants (to P. A. P. and M. R. J.), an NIH institutional postdoctoral training grant and National Research Service Award (to L. P.), an American Cancer Society, California Division, senior postdoctoral fellowship (to L. A. K.), an Arthritis Foundation fellowship (to L. T.), and an NIH Fogarty international fellowship (to M.-P. S.). 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:

MHC

major histocompatibility complex

CD-M6PR and CI-M6PR

cation-dependent and -independent mannose 6-phosphate receptor, respectively

LIMP

lysosomal integral membrane protein

PBS

phosphate-buffered saline

TGN

trans-Golgi network.
↵²L. Pond, unpublished results.
↵³L. Pond, unpublished observation.
- Received March 1, 1995.
- Revision received June 8, 1995.