Interaction of INPP5E with ARL13B is essential for its ciliary membrane retention but dispensable for its ciliary entry

Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.

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Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

Journal of Virology ◽

10.1128/jvi.02000-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 20

Author(s):

Jolene Ramsey ◽

Emily C. Renzi ◽

Randy J. Arnold ◽

Jonathan C. Trinidad ◽

Suchetana Mukhopadhyay

Keyword(s):

Plasma Membrane ◽

Sindbis Virus ◽

Molecular Mechanisms ◽

Wild Type Virus ◽

Membrane Localization ◽

Clear Understanding ◽

Wild Type ◽

Plasma Membrane Localization ◽

C Terminus ◽

N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

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Calcium Signaling Regulates Trafficking of Familial Hypocalciuric Hypercalcemia (FHH) Mutants of the Calcium Sensing Receptor

Molecular Endocrinology ◽

10.1210/me.2012-1232 ◽

2012 ◽

Vol 26 (12) ◽

pp. 2081-2091 ◽

Cited By ~ 14

Author(s):

Michael P. Grant ◽

Ann Stepanchick ◽

Gerda E. Breitwieser

Keyword(s):

Plasma Membrane ◽

Steady State ◽

State Level ◽

Membrane Targeting ◽

Familial Hypocalciuric Hypercalcemia ◽

Loss Of Function ◽

Wild Type ◽

Calcium Sensing Receptor ◽

Dynamic Changes ◽

Calcium Sensing

Abstract Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane.

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Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction

Biochemical Journal ◽

10.1042/bj20080867 ◽

2009 ◽

Vol 418 (1) ◽

pp. 163-172 ◽

Cited By ~ 49

Author(s):

Audrey Parent ◽

Emilie Hamelin ◽

Pascale Germain ◽

Jean-Luc Parent

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Glutathione Transferase ◽

Direct Interaction ◽

Small Gtpase ◽

Cell Surface Receptors ◽

Wild Type ◽

Surface Receptors ◽

Early Endosomes ◽

Intracellular Domains

The β2ARs (β2-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a β2AR–Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His6-tagged Rab11 protein and β2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of β2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the β2AR interacts preferentially with the GDP-bound form of Rab11. Arg333 and Lys348 in the C-terminal tail of the β2AR were identified as crucial determinants for Rab11 binding. A β2AR construct with these two residues mutated to alanine, β2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of β2AR RK/AA was drastically reduced when compared with wild-type β2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the β2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the β2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.

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The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers

The Journal of Cell Biology ◽

10.1083/jcb.151.4.919 ◽

2000 ◽

Vol 151 (4) ◽

pp. 919-930 ◽

Cited By ~ 126

Author(s):

E.J. Kamsteeg ◽

I. Heijnen ◽

C.H. van Os ◽

P.M.T. Deen

Keyword(s):

Plasma Membrane ◽

Steady State ◽

Subcellular Localization ◽

Water Channel ◽

Apical Plasma Membrane ◽

Wild Type ◽

Principal Cells ◽

Intracellular Vesicles ◽

And Function ◽

Fine Tune

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non–p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.

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Dual functional significance of calcineurin homologous protein 1 binding to Na+/H+ exchanger isoform 1

AJP Cell Physiology ◽

10.1152/ajpcell.00404.2010 ◽

2011 ◽

Vol 301 (2) ◽

pp. C280-C288 ◽

Cited By ~ 18

Author(s):

Masafumi Matsushita ◽

Hiroo Tanaka ◽

Keiji Mitsui ◽

Hiroshi Kanazawa

Keyword(s):

Plasma Membrane ◽

Binding Site ◽

Functional Significance ◽

Gene Knockout ◽

Membrane Localization ◽

Wild Type ◽

Homologous Protein ◽

Plasma Membrane Localization ◽

Dual Functional ◽

Associated Proteins

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na+/H+ exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246–C254, 2007). However, Pang et al. ( J Biol Chem 276: 17367–17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.

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Palmitoylation of the oncogenic RhoGEF TGAT is dispensable for membrane localization and consequent activation of RhoA

10.1101/062729 ◽

2016 ◽

Author(s):

Jakobus van Unen ◽

Dennis Botman ◽

Taofei Yin ◽

Yi I. Wu ◽

Mark A. Hink ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Single Cells ◽

Subcellular Location ◽

Membrane Localization ◽

Membrane Association ◽

Fluorescence Signal ◽

Wild Type ◽

Cellular Cytoskeleton ◽

And Control

AbstractRho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby regulate and control processes such as cell migration, cell adhesion and proliferation. TGAT is a splice variant of the RhoGEF Trio, with oncogenic potential. Whether the subcellular location of TGAT is critical for its activity is unknown. Confocal microscopy of fluorescent protein tagged TGAT revealed co-localization with a Golgi marker. Because plasma membrane localized RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was studied. In order to quantitatively measure plasma membrane association we developed a novel, highly sensitive image analysis method. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. The functional role of membrane localization was determined by measuring TGAT activity in single cells with a RhoA FRET-sensor and F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, these results show that membrane association of TGAT is critical for its activity, but that palmitoylation is dispensable.

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Protein 4.2 interaction with hereditary spherocytosis mutants of the cytoplasmic domain of human anion exchanger 1

Biochemical Journal ◽

10.1042/bj20101375 ◽

2010 ◽

Vol 433 (2) ◽

pp. 313-322 ◽

Cited By ~ 11

Author(s):

Susan P. Bustos ◽

Reinhart A. F. Reithmeier

Keyword(s):

Plasma Membrane ◽

Anion Exchanger ◽

Hereditary Spherocytosis ◽

Cytoplasmic Domain ◽

Membrane Localization ◽

Wild Type ◽

Anion Exchanger 1 ◽

Plasma Membrane Localization ◽

293 Cells ◽

Protein 4.2

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.

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