An N-terminal dileucine motif directs two-pore channels to the tonoplast of plant cells

Two‐pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C‐terminus was not essential for targeting but for channel function, while further C‐terminal truncations of two or more transmembrane domains impaired protein trafficking. An N‐terminal dileucine motif (EDPLI) proved to be critical for vacuolar targeting of TPC1, which was independent of the adaptor protein AP‐3. Deletion or mutation of this sorting motif, which is conserved among TPCs caused redirection of the protein transport to the plasma membrane. An N‐terminal region with a predicted α‐helical structure was shown to support efficient vacuolar trafficking and was essential for TPC1 function. Similar to their localization in mammalian endosomes and lysosomes, MmTPC1 and MmTPC2 were targeted to small organelles and the membrane of the lytic vacuole, respectively, when expressed in plant cells. These results shed new light on the largely uncharacterized sorting signals of plant tonoplast proteins and reveal similarities between the targeting machinery of plants and mammals.     

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.     

Positive allosteric modulation of alpha‐7 nicotinic receptors promotes cell death by inducing Ca2+ release from the endoplasmic reticulum

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7‐nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca²⁺ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca²⁺ signaling and cell viability. We used human SH‐SY5Y neuroblastoma cells overexpressing α7‐nAChRs (α7‐SH) and their control (C‐SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca²⁺ with Fura‐2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch‐clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7‐SH but not of C‐SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca²⁺ concentration; (iii) released Ca²⁺ from the ER by a Ca²⁺‐induced Ca²⁺ release mechanism only in α7‐SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7‐nAChRs, ryanodine receptors, or IP₃ receptors. In conclusion, positive allosteric modulation of α7‐nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca²⁺, overloading of intracellular Ca²⁺, and neuronal cell death.

     

Characterization of a targeting motif for a flagellar membrane protein in Leishmania enriettii.

The surface membranes of eukaryotic flagella and cilia are contiguous with the plasma membrane. Despite the absence of obvious physical structures that could form a barrier between the two membrane domains, the lipid and protein compositions of flagella and cilia are distinct from the rest of the cell surface membrane. We have exploited a flagellar glucose transporter from the parasitic protozoan Leishmania enriettii as a model system to characterize the first targeting motif for a flagellar membrane protein in any eukaryotic organism. In this study, we demonstrate that the flagellar membrane-targeting motif is recognized by several species of Leishmania. Previously, we demonstrated that the 130 amino acid NH(2)-terminal cytoplasmic domain of isoform 1 glucose transporter was sufficient to target a nonflagellar integral membrane protein into the flagellar membrane. We have now determined that an essential flagellar targeting signal is located between amino acids 20 and 35 of the NH(2)-terminal domain. We have further analyzed the role of specific amino acids in this region by alanine replacement mutagenesis and determined that single amino acid substitutions did not abrogate targeting to the flagellar membrane. However, individual mutations located within a cluster of five contiguous amino acids, RTGTT, conferred differences in the degree of targeting to the flagellar membrane and the flagellar pocket, implying a role for these residues in the mechanism of flagellar trafficking.     

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl⁻/ exchange and the failure of proton (H⁺) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.     

Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.     

Specific Interactions Between gC1qR and α1‐Adrenoceptor Subtypes

Abstract

The multi‐functional protein gC1qR has been reported to interact with an arginine‐rich motif in the C‐tail of hamster α_1B‐adrenoceptors (ARs), controlling their expression and subcellular localization. Since a similar motif is present in α_1D‐, but not α_1A‐ARs, we studied the specificity of this interaction. Human α₁‐ARs, tagged at their amino termini with Flag epitopes, were coexpressed in HEK293 cells with gC1qR containing a hemaglutinin (HA) tag at its carboxy terminus. Immunoprecipitation studies showed that Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, caused coimmunoprecipitation of HA‐gC1qR, while immunoprecipitation of HA‐gC1qR caused coimmunoprecipitation of Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, supporting specific interactions between subtypes. C‐terminal truncation of Flag‐α₁‐ARs prevented interaction with HA‐gC1qR, supporting previous conclusions about the role of the C‐terminal arginine‐rich motif. These studies suggest that gC1qR interacts specifically with α_1B‐ and α_1D‐, but not α_1A‐ARs, and this interaction depends on the presence of an intact C‐tail.     

RGD-containing ankyrin externalized onto the cell surface triggers αVβ3 integrin-mediated erythrophagocytosis

The RGD motif on the extracellular matrix or cell surface, together with its integrin receptors, constitutes a major recognition system for cell adhesion. There are several erythrocyte major membrane skeletal proteins, e.g., α spectrin, ankyrin, and protein 4.2, that bear an RGD motif. However, it is not known whether the RGD/integrin recognition system is utilized in the erythrocyte-macrophage adhesion during erythrophagocytosis. Here we report that the RGD motif of ankyrin, but not others, is recognized by the α_vβ₃ integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/or α_vβ₃. Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.     

Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins

Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca²⁺‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca²⁺‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca²⁺‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.      

Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane

The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.     

Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP – a protein that controls the disassembly of cis‐SNARE complexes – is phosphorylated. Here, we show that this post‐translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non‐phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore‐unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites.     

Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells

Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E²⁹⁵EVEALI³⁰¹, which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L³⁰⁰ and I³⁰¹ residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E²⁹⁵, E²⁹⁶, and E²⁹⁸ residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.     

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.     

Synaptojanin 1 Is Required for Endolysosomal Trafficking of Synaptic Proteins in Cone Photoreceptor Inner Segments

Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc^a14, which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc^a14 cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc^a14 inner segment. The structure of the Endoplasmic Reticulum in nrc^a14 mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc^a14 cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc ^a14 mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.     

Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p

Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.