Myosin 1G is an abundant class I myosin in lymphocytes whose localization at the plasma membrane depends on its ancient divergent pleckstrin homology (PH) domain (Myo1PH)

Abstract

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a "Pre-PH" region; and 3) a "Post-PH" region. The Pre-PH predicted alpha helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the "signature motif" destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the beta3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.

FIGURE 1.

Myosins identified in the fractions of human PBT and 300.19. Quantitation of the number of peptides identified from each myosin gene in mass spectrometric analysis of membrane/microvillus (MMV, filled bars) fraction versus post nuclear lysate (PNL, open bars) of two lymphoid cells: human PBT (A) and a mouse pre B cell line (300.19) (B). Conservative criteria were used for identifying each protein (namely detection of two distinct peptides from that protein using identification thresholds that gave a 5% false positive for any single peptide).

FIGURE 2.

Characterization of Myo1G expression by Western blot. A, WB of Myo1G in whole cell lysate (60 μg) from the indicated cell types with WB for actin as loading control; see supplemental Fig. S1 for entire molecular weight range of this WB; B, WB of Myo1G from purified mouse T- and B-lymphocytes with WB for moesin as loading control.

FIGURE 3.

Localization of Myo1G at the plasma membrane. A, immunofluorescence analysis of fixed and permeabilized PBT (top), Jurkat (middle), and 300.19 (bottom) stained with rabbit serum anti Myo1G followed by secondary antibody conjugated to Alexa 488. A representative mid plane image (left panel) and projection image (right panel) are shown. Bars, 5 μm. B, WB of Myo1G in 1% Triton X-100 lysate, in the absence or presence of ATP. WB of cytoskeletal regulating cytosolic protein WIP is shown as control. C, mid plane immunofluorescence images of live cells transfected with full length GFP-Myo1G fusion protein: i, PBT; ii, 300.19; iii, Jurkat; and iv, HeLa. Bars, 5 μm.

FIGURE 4.

IQ plus tail regions of Myo1G, Myo1C, and Myo1F are sufficient for membrane association. A, schematic representation of short tail (Myo1G and Myo1C) and long tail (Myo1F) class I myosin constructs studied. B, representative immunofluorescence images of Jurkat cells expressing constructs of Myo1G or Myo1C. Bar, 5 μm. C, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line). D, representative immunofluorescence images of Jurkat cells expressing Myo1F constructs. Bar, 5 μm. E, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line).

FIGURE 5.

Fine mapping of the “minimal” region of Myo1G tail for membrane localization. A, schematic representation of Myo1G fragments fused with GFP for membrane localization studies. The region shown is Myo1G C-terminal to the motor domain. The regions marked on the map include: IQ motifs, boundaries of the TH1 region identified by PFAM motif PF0617 (dashed line), the Myo1PH domain corresponding to PH-like domain previously described (14) and identified by our bioinformatic analysis, and the Pre-PH and Post-PH regions identified by our functional analysis. The boundaries of the constructs tested are shown relative to these regions. An arrowhead pointing to the left indicates that the construct includes the motor domain. B, representative mid plane images of Jurkat cells transfected with the indicated constructs. Bar, 5 μm. C, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line).

FIGURE 6.

Functional effect of mutating positively charged residues in the third predicted α helix in the Pre-PH region of Myo1G. A, helical wheel representation of the third predicted helix in the Pre-PH region of Myo1G. Arcs represent predicted hydrophobic surfaces. Positively charged residues are annotated with blue numbers indicating the number of human short tail myosins in which the residue is conserved. B, representative images of Jurkat cells transfected with the indicated point mutants in the Pre-PH of Myo1G; C, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line).

FIGURE 7.

Multiple sequence alignment of the Myo1PH domain of representative proteins identified by the Myo1PH sequence profile. A Myo1PH sequence profile was developed and used to screen the non-redundant (nr) sequence data base as described under “Experimental Procedures.” Shown here is the Myo1PH region of an informative panel of the protein hits, including all human class I myosins, a yeast long tail myosin, proteins from diverse eukaryotes, and the bacterial protein. For expansion of information on organism and listing of GI number see supplemental Table S1. In the histogram shown below the alignment represents ClustalX calculation of relative conservation at each position. The schematic above the alignment shows secondary structure predictions, which are concordant with those observed in the classic PH domain. The blue asterisks represent all basic residues in Myo1G, each of which was mutated to assess function. The red arrowheads indicate the three residues whose mutation impairs membrane localization. 1 (Lys-877) and 2 (Arg-887) are the signature residues in the β1/2 loop. 3 (Lys-898) in β3 strand was first identified as being important in our mutational screen.

FIGURE 8.

Identification of Arg and Lys residues in the Myo1PH that contribute to membrane localization. A, Jurkat cells were transfected with point mutants of the “signature residues” in the β1/β2 loop of the PH domain. B, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line). C, Jurkat cells transfected with point mutants in all the other Arg and Lys residues within “Myo1PH.” D, quantitative analysis of membrane enrichment for the indicated mutants. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line).

FIGURE 9.

Effect of acute PIP₂ reduction at the plasma membrane on localization of Myo1G. Jurkat cells were transfected with a rapamycin-inducible system to acutely reduce PIP₂ levels in the plasma membrane (see “Experimental Procedures”). A, representative single-color images of individual cells transfected with the membrane-targeting construct (FRB-CFP, blue); the 5-Ptase construct (mRFP-FKBP-5-Ptase, red), and either Myo1G or Myo1C (green) in the presence or absence of Rapamycin. B, quantitative analysis of membrane enrichment for the indicated conditions. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization in the range from 0.5 to 1.0 (dashed line).