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Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor.

Fueller J, Egorov MV, Walther KA, Sabet O, Mallah J, Grabenbauer M, Kinkhabwala A - PLoS ONE (2015)

Bottom Line: Here, we show that PTP1B's mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor.Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor.Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.

View Article: PubMed Central - PubMed

Affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany; Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.

ABSTRACT
The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B's mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B's insertion into the ER membrane through heterologous expression of PTP1B's tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.

No MeSH data available.


Tail anchor targeting is largely dictated by three different factors: TMD length, C-terminal charge and hydropathy.Our experimental localization results for different tail isoforms in COS-7 cells and yeast cells are summarized. We have shown that the wild-type localization of PTP1Btail to the mitochondria and ER (Mito/ER) is highly sensitive to changes in these three factors (see text and S8 Fig, S9 Fig and S10 Fig).
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pone.0139429.g006: Tail anchor targeting is largely dictated by three different factors: TMD length, C-terminal charge and hydropathy.Our experimental localization results for different tail isoforms in COS-7 cells and yeast cells are summarized. We have shown that the wild-type localization of PTP1Btail to the mitochondria and ER (Mito/ER) is highly sensitive to changes in these three factors (see text and S8 Fig, S9 Fig and S10 Fig).

Mentions: In the above, we have shown that the subcellular targeting of PTP1B depends sensitively on three generic features of its tail anchor—namely, its TMD length, charge and hydropathy. Our overall findings regarding the localization(s) of the various isoforms that we tested in both mammalian and yeast cells are summarized in Fig 6 and S1 Table. The most significant result is the clear dependence on charge of ER versus mitochondrial partitioning in both mammalian and yeast cells, with negative C-terminal charge leading to complete retention to the ER and (increasingly) positive charge shifting the balance to the mitochondria. That this charge dependence is similar in yeast importantly suggests evolutionarily shared mechanisms for delivery of PTP1B (or closely related isoforms) to the mitochondria. No mammalian-specific chaperones are therefore required to explain the mitochondrial localization of the wild-type isoform in mammalian cells but not yeast. Instead, slight differences in the charged lipid composition of these organelles in mammalian versus yeast cells provides the most compelling explanation. In addition to charge, it is clear that tail anchor hydropathy is another important factor that specifically impacts the ER-mitochondrial balance. Mutation of a single amino acid, leading to an increased hydropathy, was sufficient to restrict PTP1B’s tail anchor to the ER in mammalian cells, suggesting that the ER membrane presents a better environment for tail anchors with high hydropathy than the mitochondrial membrane (in line with the “hydropathic code”, S5 Fig). Both charge and hydropathy are therefore independently capable of affecting the ER-to-mitochondrial balance. Finally, the TMD length of the tail anchor introduces yet another important variable; however, the more complex results from these studies (which differ in mammalian cells versus yeast) do not provide as straightforward an interpretation as changes in charge and hydropathy.


Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor.

Fueller J, Egorov MV, Walther KA, Sabet O, Mallah J, Grabenbauer M, Kinkhabwala A - PLoS ONE (2015)

Tail anchor targeting is largely dictated by three different factors: TMD length, C-terminal charge and hydropathy.Our experimental localization results for different tail isoforms in COS-7 cells and yeast cells are summarized. We have shown that the wild-type localization of PTP1Btail to the mitochondria and ER (Mito/ER) is highly sensitive to changes in these three factors (see text and S8 Fig, S9 Fig and S10 Fig).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592070&req=5

pone.0139429.g006: Tail anchor targeting is largely dictated by three different factors: TMD length, C-terminal charge and hydropathy.Our experimental localization results for different tail isoforms in COS-7 cells and yeast cells are summarized. We have shown that the wild-type localization of PTP1Btail to the mitochondria and ER (Mito/ER) is highly sensitive to changes in these three factors (see text and S8 Fig, S9 Fig and S10 Fig).
Mentions: In the above, we have shown that the subcellular targeting of PTP1B depends sensitively on three generic features of its tail anchor—namely, its TMD length, charge and hydropathy. Our overall findings regarding the localization(s) of the various isoforms that we tested in both mammalian and yeast cells are summarized in Fig 6 and S1 Table. The most significant result is the clear dependence on charge of ER versus mitochondrial partitioning in both mammalian and yeast cells, with negative C-terminal charge leading to complete retention to the ER and (increasingly) positive charge shifting the balance to the mitochondria. That this charge dependence is similar in yeast importantly suggests evolutionarily shared mechanisms for delivery of PTP1B (or closely related isoforms) to the mitochondria. No mammalian-specific chaperones are therefore required to explain the mitochondrial localization of the wild-type isoform in mammalian cells but not yeast. Instead, slight differences in the charged lipid composition of these organelles in mammalian versus yeast cells provides the most compelling explanation. In addition to charge, it is clear that tail anchor hydropathy is another important factor that specifically impacts the ER-mitochondrial balance. Mutation of a single amino acid, leading to an increased hydropathy, was sufficient to restrict PTP1B’s tail anchor to the ER in mammalian cells, suggesting that the ER membrane presents a better environment for tail anchors with high hydropathy than the mitochondrial membrane (in line with the “hydropathic code”, S5 Fig). Both charge and hydropathy are therefore independently capable of affecting the ER-to-mitochondrial balance. Finally, the TMD length of the tail anchor introduces yet another important variable; however, the more complex results from these studies (which differ in mammalian cells versus yeast) do not provide as straightforward an interpretation as changes in charge and hydropathy.

Bottom Line: Here, we show that PTP1B's mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor.Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor.Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.

View Article: PubMed Central - PubMed

Affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany; Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.

ABSTRACT
The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B's mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B's insertion into the ER membrane through heterologous expression of PTP1B's tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.

No MeSH data available.