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Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

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Palmitoylation of GAP43 is required for membrane association. (A) Images of single focal planes of living PC12 cells expressing different PAGFP-tagged palmitoylation-deficient constructs (GAP43C3,4S) as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z-stack images. Mean ± SEM, n = 13–20 cells from two independent experiments. ###Significantly different values compared with GAP43wt or the respective phosphomutants without the C3,4S mutation (Figure 2A, right). Note the complete absence of peripheral enrichment in all palmitoylation-negative mutants. Scale bar, 10 μm. (B) Quantitation of the palmitoylation state of GAP43 and the two phosphomutants by [3H]palmitate labeling. The value for GAP43wt was set as 100%. Experiments were conducted two times with three biological repetitions. Mean ± SEM, three to five independent experiments. (C) Subcellular fractionation of PC12 cells that have been lentivirally transduced to express the different PAGFP-tagged GAP43 constructs as indicated. Immunoblot showing membrane (m) and cytosolic (c) fractions (top) and the respective quantitation (bottom). Blots were developed using anti-GFP antibody. Quantitation was performed by determining the ratio of the membrane (m) to total (m + c) signal after densitometric analysis. The value for GAP43wt was set as 1.0. Mean ± SEM from three to five independent experiments. Numbers to the side of the gel blot indicate molecular mass standards in kilodaltons. Statistical analysis was performed using Student's t test. p values are as follows: *, p < 0.05 ***(###), p < 0.001.
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Figure 3: Palmitoylation of GAP43 is required for membrane association. (A) Images of single focal planes of living PC12 cells expressing different PAGFP-tagged palmitoylation-deficient constructs (GAP43C3,4S) as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z-stack images. Mean ± SEM, n = 13–20 cells from two independent experiments. ###Significantly different values compared with GAP43wt or the respective phosphomutants without the C3,4S mutation (Figure 2A, right). Note the complete absence of peripheral enrichment in all palmitoylation-negative mutants. Scale bar, 10 μm. (B) Quantitation of the palmitoylation state of GAP43 and the two phosphomutants by [3H]palmitate labeling. The value for GAP43wt was set as 100%. Experiments were conducted two times with three biological repetitions. Mean ± SEM, three to five independent experiments. (C) Subcellular fractionation of PC12 cells that have been lentivirally transduced to express the different PAGFP-tagged GAP43 constructs as indicated. Immunoblot showing membrane (m) and cytosolic (c) fractions (top) and the respective quantitation (bottom). Blots were developed using anti-GFP antibody. Quantitation was performed by determining the ratio of the membrane (m) to total (m + c) signal after densitometric analysis. The value for GAP43wt was set as 1.0. Mean ± SEM from three to five independent experiments. Numbers to the side of the gel blot indicate molecular mass standards in kilodaltons. Statistical analysis was performed using Student's t test. p values are as follows: *, p < 0.05 ***(###), p < 0.001.

Mentions: To determine whether phosphorylation of GAP43 at Ser-41 is sufficient to induce plasma membrane association, we created a palmitoylation-deficient GAP43 construct by mutating the two cysteine residues at positions 3 and 4 to serine (GAP43C3,4S; Liu et al., 1994) and also as a combination with the two phosphomutations at Ser-41. None of the three constructs showed detectable enrichment at the cell periphery in living cells (Figure 3A, left). Quantification by densitometric analysis confirmed the absence of peripheral enrichment (Figure 3A, right), indicating an absolute requirement for palmitoylation for plasma membrane association. The fact that all palmitoylation-deficient GAP43 constructs exhibited a significant lower peripheral enrichment than the respective palmitoylatable counterparts indicates that palmitoylation alone mediates some, albeit apparently weak, association with the plasma membrane, which is increased after phosphorylation. This is also detectable in the images of the single focal plane of the respective nonphosphorylatable but palmitoylatable GAP43S41A-expressing cells (e.g., arrows in Figure 2A).


Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Palmitoylation of GAP43 is required for membrane association. (A) Images of single focal planes of living PC12 cells expressing different PAGFP-tagged palmitoylation-deficient constructs (GAP43C3,4S) as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z-stack images. Mean ± SEM, n = 13–20 cells from two independent experiments. ###Significantly different values compared with GAP43wt or the respective phosphomutants without the C3,4S mutation (Figure 2A, right). Note the complete absence of peripheral enrichment in all palmitoylation-negative mutants. Scale bar, 10 μm. (B) Quantitation of the palmitoylation state of GAP43 and the two phosphomutants by [3H]palmitate labeling. The value for GAP43wt was set as 100%. Experiments were conducted two times with three biological repetitions. Mean ± SEM, three to five independent experiments. (C) Subcellular fractionation of PC12 cells that have been lentivirally transduced to express the different PAGFP-tagged GAP43 constructs as indicated. Immunoblot showing membrane (m) and cytosolic (c) fractions (top) and the respective quantitation (bottom). Blots were developed using anti-GFP antibody. Quantitation was performed by determining the ratio of the membrane (m) to total (m + c) signal after densitometric analysis. The value for GAP43wt was set as 1.0. Mean ± SEM from three to five independent experiments. Numbers to the side of the gel blot indicate molecular mass standards in kilodaltons. Statistical analysis was performed using Student's t test. p values are as follows: *, p < 0.05 ***(###), p < 0.001.
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Figure 3: Palmitoylation of GAP43 is required for membrane association. (A) Images of single focal planes of living PC12 cells expressing different PAGFP-tagged palmitoylation-deficient constructs (GAP43C3,4S) as indicated. Photoactivation was performed in the whole field of view before imaging. Right, quantitation of peripheral enrichment by densitometric analysis of single z-stack images. Mean ± SEM, n = 13–20 cells from two independent experiments. ###Significantly different values compared with GAP43wt or the respective phosphomutants without the C3,4S mutation (Figure 2A, right). Note the complete absence of peripheral enrichment in all palmitoylation-negative mutants. Scale bar, 10 μm. (B) Quantitation of the palmitoylation state of GAP43 and the two phosphomutants by [3H]palmitate labeling. The value for GAP43wt was set as 100%. Experiments were conducted two times with three biological repetitions. Mean ± SEM, three to five independent experiments. (C) Subcellular fractionation of PC12 cells that have been lentivirally transduced to express the different PAGFP-tagged GAP43 constructs as indicated. Immunoblot showing membrane (m) and cytosolic (c) fractions (top) and the respective quantitation (bottom). Blots were developed using anti-GFP antibody. Quantitation was performed by determining the ratio of the membrane (m) to total (m + c) signal after densitometric analysis. The value for GAP43wt was set as 1.0. Mean ± SEM from three to five independent experiments. Numbers to the side of the gel blot indicate molecular mass standards in kilodaltons. Statistical analysis was performed using Student's t test. p values are as follows: *, p < 0.05 ***(###), p < 0.001.
Mentions: To determine whether phosphorylation of GAP43 at Ser-41 is sufficient to induce plasma membrane association, we created a palmitoylation-deficient GAP43 construct by mutating the two cysteine residues at positions 3 and 4 to serine (GAP43C3,4S; Liu et al., 1994) and also as a combination with the two phosphomutations at Ser-41. None of the three constructs showed detectable enrichment at the cell periphery in living cells (Figure 3A, left). Quantification by densitometric analysis confirmed the absence of peripheral enrichment (Figure 3A, right), indicating an absolute requirement for palmitoylation for plasma membrane association. The fact that all palmitoylation-deficient GAP43 constructs exhibited a significant lower peripheral enrichment than the respective palmitoylatable counterparts indicates that palmitoylation alone mediates some, albeit apparently weak, association with the plasma membrane, which is increased after phosphorylation. This is also detectable in the images of the single focal plane of the respective nonphosphorylatable but palmitoylatable GAP43S41A-expressing cells (e.g., arrows in Figure 2A).

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

Show MeSH
Related in: MedlinePlus