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, 52 (10), 601-606

Deciphering the Molecular Mechanisms Underlying the Plasma Membrane Targeting of PRMT8

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Deciphering the Molecular Mechanisms Underlying the Plasma Membrane Targeting of PRMT8

Sang-Won Park et al. BMB Rep.

Abstract

Arginine methylation plays crucial roles in many cellular functions including signal transduction, RNA transcription, and regulation of gene expression. Protein arginine methyltransferase 8 (PRMT8), a unique brain-specific protein, is localized to the plasma membrane. However, the detailed molecular mechanisms underlying PRMT8 plasma membrane targeting remain unclear. Here, we demonstrate that the N-terminal 20 amino acids of PRMT8 are sufficient for plasma membrane localization and that oligomerization enhances membrane localization. The basic amino acids, combined with myristoylation within the N-terminal 20 amino acids of PRMT8, are critical for plasma membrane targeting. We also found that substituting Gly-2 with Ala [PRMT8(G2A)] or Cys-9 with Ser [PRMT8(C9S)] induces the formation of punctate structures in the cytosol or patch-like plasma membrane localization, respectively. Impairment of PRMT8 oligomerization/dimerization by Cterminal deletion induces PRMT8 mis-localization to the mitochondria, prevents the formation of punctate structures by PRMT8(G2A), and inhibits PRMT8(C9S) patch-like plasma membrane localization. Overall, these results suggest that oligomerization/dimerization plays several roles in inducing the efficient and specific plasma membrane localization of PRMT8. [BMB Reports 2019; 52(10): 601-606].

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Plasma membrane targeting of PRMT8-GFP. (A) Schematic diagram of PRMT8 wild-type (PRMT8-GFP) and serial mutants. (B, C) Cellular localization of PRMT8 serial deletion mutants. PRMT8-GFP, PRMT8(ΔN15)-GFP, PRMT8(N270)-GFP, PRMT8(N220)-GFP, PRMT8(N60)-GFP, and PRMT8(N20)-GFP localized to the plasma membrane in HEK293T cells (B) and in cultured cortical neurons (C). Scale bar, 20 μm. SH3BD, SH3-binding domain. (D) Quantification of the ratio between the fluorescent intensity at the plasma membrane and in the cytosol of cells expressing the PRMT8 constructs in HEK293T cells. *P < 0.001, one-way ANOVA; F = 12.96, Tukey’s post-hoc test. Values are presented as means ± SEM. Scale bar, 20 μm. (E) Oligomerization/dimerization of PRMT8. PRMT8-3×FLAG was co-expressed with PRMT8-GFP, PRMT8(N270)-GFP, PRMT8(N220)-GFP, PRMT8(N60)-GFP, or GFP in HEK293T cells. The data shown represent the results from three independent experiments. 1% of total lysate was used as input. (F) Quantification of the relative interaction of PRMT8-3xFLAG to PRMT8, PRMT8(ΔN15), PRMT8(N270), and PRMT8(N220)-GFP. ***P < 0.0001, one-way ANOVA; F = 69.36, Tukey’s post-hoc test. Values are presented as means ± SEM. N. S., not significant.
Fig. 2
Fig. 2
Characterization of the PRMT8 plasma membrane-targeting domains. (A) Schematic diagrams of point mutations within the N-terminal 20 amino acids of PRMT8. Mutated amino acids are underlined. Basic amino acids are colored in red. (B, C) Cellular localization of PRMT8-GFP, PRMT8(G2A)-GFP, and PRMT8(K4,15A/R6,13,14A)-GFP in HEK293T cells (B) and in cultured cortical neurons (C) Scale bar, 20 μm. (D) Cellular localization of PRMT8(G2A)-GFP, PRMT8(G2A/N270)-GFP, and PRMT8(G2A/N20)-GFP in HEK293T cells. Scale bar, 20 μm. (E) Effects of phosphoinositide depletion on the plasma membrane localization of PRMT8-GFP and Aplysia PDE4 short-form, S(N-UCR1/2)-GFP after antimycin treatment. Images were acquired before and after treatment with 10 μM antimycin for 40 min. Scale bar, 20 μm. (F) Cellular localization of PRMT8(C9S)-GFP and PRMT8 (C9S/N20)-GFP in HEK293T cells. Scale bar, 20 μm.
Fig. 3
Fig. 3
Mitochondria mis-targeting of PRMT8 mutants. (A) Mitochondrial localization of PRMT8 mutants. PRMT8-GFP, PRMT8(N270)-GFP, and PRMT8(N20)-GFP co-localized with Tom20-mRFP, a mitochondria marker, in HEK293T cells. Scale bar, 20 μm. (B) Mitochondrial luminal localization of PRMT8(N20)-GFP in HEK293T cells. PRMT8(N20)-GFP co-localized with Tom20-mRFP, a mitochondrial outer membrane marker. The yellow line in the confocal fluorescence images indicates the paths along which the fluorescence intensities of the corresponding images were plotted to the bottom [red line: Tom20-mRFP, green line: PRMT8(N20)-GFP]. Scale bar, 20 μm. (C) N-terminal 15 amino acid sequences of PRMT8 within the amino acids. +, basic amino acids; H, hydroxylated amino acids; O, hydrophobic amino acids; mG, myristoylated Gly. (D) Cellular localization of PRMT8(N20/G2A)-GFP or PRMT8(N20/K4,15A,R6,13,14A)-GFP. Mitochondria are stained with MitoTracker, a mitochondria marker. The yellow line in the confocal fluorescence images indicates the paths along which the fluorescence intensities of the corresponding images were plotted to the bottom [red line: MitoTracker; green line: PRMT8(N20/K4,15A,R6,13,14A)-GFP]. Scale bar, 20 μm.

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