In Vitro Fusion of Phagosomes with Different Endocytic Organelles from J774 Macrophages

doi:10.1074/jbc.273.46.30379

In Vitro Fusion of Phagosomes with Different Endocytic Organelles from J774 Macrophages*

From the ^‡Cell Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, D-69012 Heidelberg, Germany, the ^¶University of Oslo, MCB, P.O. Box 1050, Blindern, 0136 Oslo, Norway, the ^‖Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061-0308, and ^**Institut für Biochemie, Universität Mainz, Becherweg 30, D-55128 Mainz, Germany

Abstract

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.

Footnotes

↵* This work was supported by a network grant from the Human frontier Science Program (to G. G.).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 in part by Deutsche Forschungsgemeinschaft Grant SFB 352.
↵‡ To whom all correspondence should be addressed: Cell Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, D-69012. Tel.: 49-6221-387267; Fax: 49-6221-387267; E-mail:griffiths{at}embl-heidelberg.de.
↵2 M. A. Shonn, A. Blocker, J. Burkhardt, G. Griffiths, D. G. Weiss, and S. Kusnetzov, manuscript in preparation.
↵3 A. Jahraus, M. Egeberg, M. Cyrklaf, A. Habermann, H. Faulstich, D. Echner, A. Pralle, H. Hörber, H. Defaque, and G. Griffiths, manuscript in preparation.
↵4 H. Defaque, M. Egeberg, A. Habermann, M. Diakonova, C. Roy, P. Mangeat, W. Voeltner, G. Marriott, J. Pfannstiel, H. Faulstich, and G. Griffiths, submitted for publication.
Abbreviations:

PNS

postnuclear supernatant extract(s)

MES

4-morpholineethanesulfonic acid

PBS

phosphate-buffered saline

IM

internalization medium

TES

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid

MOPS

4-morpholinepropanesulfonic acid

HRP

horseradish peroxidase

bHRP

biotinylated HRP

BSA

bovine serum albumin

GTPγS

guanosine 5′-3-O-(thio)triphosphate

EM

electron microscopy

GDI

GDP dissociation inhibitor

HB

homogenization buffer.
- Received May 27, 1998.
- Revision received August 3, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.