Plasticity of PI4KIIIα interactions at the plasma membrane

Abstract

Plasma membrane PI4P is an important direct regulator of many processes that occur at the plasma membrane and also a biosynthetic precursor of PI(4,5)P2 and its downstream metabolites. The majority of this PI4P pool is synthesized by an evolutionarily conserved complex, which has as its core the PI 4-kinase PI4KIIIα (Stt4 in yeast) and also comprises TTC7 (Ypp1 in yeast) and the peripheral plasma membrane protein EFR3. While EFR3 has been implicated in the recruitment of PI4KIIIα via TTC7, the plasma membrane protein Sfk1 was also shown to participate in this targeting and activity in yeast. Here, we identify a member of the TMEM150 family as a functional homologue of Sfk1 in mammalian cells and demonstrate a role for this protein in the homeostatic regulation of PI(4,5)P2 at the plasma membrane. We also show that the presence of TMEM150A strongly reduces the association of TTC7 with the EFR3-PI4KIIIα complex, without impairing the localization of PI4KIIIα at the plasma membrane. Collectively our results suggest a plasticity of the molecular interactions that control PI4KIIIα localization and function.

Keywords: PI4KA; Rolling blackout; Ypp1; phospholipase C.

Figure 1

Identification of a mammalian homologue of yeast Sfk1

1. A Domain cartoons of yeast Sfk1 and of its putative homologues in mammals. The light gray portions represent FRAG1/DRAM/Sfk1 family homology and the superimposed darker boxes represent transmembrane domain regions.

2. B Confocal imaging of live HeLa cells transfected with TMEM150A-GFP, TMEM150B-GFP, and TMEM150C-GFP as indicated. Scale bars: 20 μm.

3. C Western blot analysis for GFP and PI4KIIIα of anti-GFP immunoprecipitates from lysates of HeLa cells transfected with GFP or TMEM150A-GFP fusion proteins.

Figure 2

TMEM150A interacts with the PI4KIIIα complex via its C-terminal tail

1. A Fluorescence of HeLa cells expressing TMEM150A-GFP (top) or HA-TMEM150A-GFP (bottom) (see cartoons at left) and incubated with anti-GFP (top) or anti-HA (bottom) antibodies without permeabilization before fixation. Left panels represent intrinsic GFP fluorescence, and right panels represent anti-GFP or anti-HA immunofluorescence.

2. B Schematic diagram of TMEM150A, TMEM150B, and Chimera. Chimera was generated by fusing the C-terminal 40 amino acids of TMEM150A (dotted box) to the C-terminus of full-length TMEM150B.

3. C Snapshot of confocal live imaging of HeLa cell expressing Chimera-GFP. Scale bar: 20 μm.

4. D Anti-FLAG and anti-GFP Western blots of immunoprecipitates generated from lysates of HeLa cells double-transfected with 3 × FLAG-PI4KIIIα and either TMEM150A-GFP, TMEM150B-GFP, or Chimera-GFP.

Figure 3

Interplay between TMEM150A and components of the PI4KIIIα complex

1. A Confocal imaging for GFP and mCherry fluorescence of live HeLa cells transfected with GFP-PI4KIIIα, TMEM150A-mCherry, EFR3B-HA, and TTC7B-FLAG in various combinations. Scale bars: 20 μm.

2. B Western blot analysis for the epitopes and proteins indicated, of starting lysates and anti-GFP immunoprecipitates generated from HeLa cells treated with control or EFR3 (EFR3A and EFR3B)-specific siRNAs 24 h prior to transfection of 3 × FLAG-PI4KIIIα and TMEM150A-GFP as shown at the top.

3. C Western blot analysis for the epitopes and proteins indicated, of starting lysates and anti-GFP immunoprecipitates generated from HeLa cells transfected with 3 × FLAG-PI4KIIIα, EFR3B-HA, TTC7B-mCherry, and TMEM150A-GFP as shown at the top.

4. D Western blot analysis for the epitopes and protein indicated, of starting lysates and anti-GFP immunoprecipitates generated from HeLa cells treated with control and PI4KIIIα-specific siRNA 24 h prior to transfection of EFR3B-HA and TMEM150A-GFP as shown at the top.

Figure 4

The interaction of the EFR3-PI4KIIIα complex with TMEM150A is mutually exclusive with the interaction with TTC7

1. A-C Confocal imaging of live HeLa cells transfected with TTC7B-mCherry alone or with EFR3B-HA and TMEM150A-GFP as indicated. Only the fluorescence of TTC7B-mCherry is shown. Scale bars: 20 μm.

2. D Anti-HA and anti-FLAG Western blots of starting lysates and immunoprecipitates generated from lysates of HeLa cells triple-transfected with EFR3B-HA, TTC7B-FLAG, and TMEM150A-GFP. Protein complexes were immunoprecipitated by anti-HA (top panels) or anti-FLAG (bottom panels) antibodies. The asterisk indicates fragments of TTC7B just below the full-length TTC7B bands.

3. E Western blot analysis for the epitopes and proteins indicated, of starting lysates and anti-GFP immunoprecipitates generated from HeLa cells transfected with 3 × FLAG-PI4KIIIα, EFR3B-HA, TMEM150A-mCherry, and TTC7B-GFP as shown at the top.

Figure 5

Levels of TMEM150A affect the rate of PI(4,5)P₂ resynthesis at the plasma membrane

1. A Time course of GFP fluorescence, as assessed by TIRF microscopy, from HeLa cells transfected with GFP-PH_PLCδ and muscarinic receptor (M1R) and pretreated with compound A1 (100 nM) for 10 min. Oxo-M (10 mM) and atropine (50 mM) were added at the indicated times. Kymographs of representative cells (top) and normalized average traces (bottom; n ≥ 10) are shown. Quantitative data are represented as mean ± SEM.

2. B Time course of GFP fluorescence as in (A) from HeLa cells expressing the indicated proteins together with GFP-PH_PLCδ and M1R.

3. C Bar graph showing initial rate of PI(4,5)P₂ recovery (before atropine) from (B), represented as mean ± SEM (n ≥ 10). Statistical significance was assessed by Student's t-test. ****P < 0.0001.

4. D Time course of normalized GFP fluorescence, as in (A) from HeLa cells expressing the indicated proteins together with GFP-PH_PLCδ and M1R, and in addition treated with control TMEM150A-specific siRNA 24 h prior to other transfection.

5. E Bar graph showing initial rate of PI(4,5)P₂ recovery (after atropine) from (D), represented as mean ± SEM (n ≥ 10). Statistical significance was assessed by Student's t-test. ****P < 0.0001.

6. F Schematic representation of PI4KIIIα interactions at the plasma membrane. TTC7 interacts directly with both PI4KIIIα and EFR3. The presence of TMEM150A in a complex comprising PI4KIIIα is mutually exclusive to the presence in the complex of TTC7. The interactions of TMEM150A shown at the right were demonstrated biochemically and result in a positive regulation of PI4KIIIα but may be indirect.