The pH-sensitive Actin-binding Protein Hisactophilin of Dictyostelium Exists in Two Isoforms Which Both Are Myristoylated and Distributed between Plasma Membrane and Cytoplasm

doi:10.1074/jbc.270.2.596

The pH-sensitive Actin-binding Protein Hisactophilin of Dictyostelium Exists in Two Isoforms Which Both Are Myristoylated and Distributed between Plasma Membrane and Cytoplasm (*)

From the Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany

Abstract

The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino acid sequence of the protein. Electrospray mass spectrometry and liquid secondary ion mass spectrometry of tryptic fragments separated by reversed-phase high performance liquid chromatography (HPLC) identified the most hydrophobic peptide as a myristoylated fragment from the N terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N terminally modified by myristoylation. By reversed-phase HPLC, two isoforms of hisactophilin, HsI and HsII, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of HsI fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristoylation considered), HsII is another protein distinguished from HsI by several amino acid exchanges. HsI and HsII can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 266/D7 (to G. G.). 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:

DTT

dithiothreitol

ES-MS

electrospray mass spectrometry

HPLC

high performance liquid chromatography

HsI

hisactophilin I

HsII

hisactophilin II

LC-MS

liquid chromatography mass spectrometry

LSI-MS

liquid secondary ion mass spectrometry

MS

mass spectrometry

Tricine

N-tris(hydroxymethyl)methylglycine

MES

4-morpholineethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis.
- Received June 14, 1994.
- Revision received October 17, 1994.