Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes

doi:10.1074/jbc.273.32.20121

Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes*

From the ^‡Department of Cell Biology, Utrecht University School of Medicine, AZU, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands and ^‖Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan

Abstract

Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50–60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by Grant ERB4050PL940675 from the European Community. Present address: Dept. of Biochemistry, University of Geneva, 30 quai E. Ansermet, 1211 Geneva 4, Switzerland.
↵¶ Recipient of Grant 901-09-241 from Nederlandse Organisatie voor Wetenschappelijk Onderzoek.
↵** To whom correspondence should be addressed. Tel.: 31-30-250-6551; Fax: 31-30-254-1797; E-mail: H.J.Geuze{at}lab.azu.nl.
↵2 M. J. Kleijmeer and H. J. Geuze, unpublished data.
Abbreviations:

MHC

major histocompatibility complex

HLA

human leukocyte antigen

IEM

immunoelectron microscopy

Ii

invariant chain

MIIC

MHC class II-enriched compartment

PAGE

polyacrylamide gel electrophoresis

TfR

transferrin receptor.
- Received January 20, 1998.
- Revision received May 18, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.