Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.
The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.
Baines
I C
IC
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Corigliano-Murphy
A
A
Korn
E D
ED
D000048:Acanthamoeba / Q000201:enzymology / Q000648:ultrastructure*; D000199:Actins / Q000302:isolation & purification; D000818:Animals; D000918:Antibody Specificity; D017301:Ca(2+) Mg(2+)-ATPase / Q000276:immunology / Q000302:isolation & purification*; D002451:Cell Compartmentation; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006046:Gold; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D007834:Lasers; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009218:Myosins / Q000276:immunology / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D014617:Vacuoles / Q000648:ultrastructure
D000199:Actins; D007527:Isoenzymes; D010750:Phosphoproteins; D006046:Gold; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins