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© The Rockefeller University Press, 0021-9525/1997//1059 $5.00
The Journal of Cell Biology, Volume 136, Number 5, , 1997 1059-1070

Isoforms of Ankyrin-3 That Lack the NH₂-terminal Repeats Associate with Mouse Macrophage Lysosomes

Thomas C. Hoock^*,, Luanne L. Peters^*, and Samuel E. Lux^*

^* Division of Hematology/Oncology, Children's Hospital and the Dana Farber Cancer Institute, Division of Medical Science, Program in Biological and Biomedical Sciences, Harvard University, Boston, Massachusetts 02115

We have recently cloned and characterized ankyrin-3 (also called ankyrin_G), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313–330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH₂-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29–amino acid segment of the membrane ("repeat") domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrin_R), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3–positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran– containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3–positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.

Abbreviations used in this paper: aa, amino acid; BMM, bone marrow macrophage medium; GST, glutathione-S-transferase; HA, hemagglutinin; IL, interleukin; LAMP-1, lysosomal-associated membrane glycoprotein 1; RACE, rapid amplification of cDNA ends; RT, reverse transcription.

L.L. Peter's present address is The Jackson Laboratory, Bar Harbor, ME 04609.

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