Reticulon-like proteins in Arabidopsis thaliana: structural organization and ER localization

Reticulons are proteins that have been found predominantly associated with the endoplasmic reticulum in yeast and mammalian cells. While their functions are still poorly understood, recent findings suggest that they participate in the shaping of the tubular endoplamic reticulum (ER). Although reticulon-like proteins have been identified in plants, very little is known about their cellular localization and functions. Here, we characterized the reticulon-like protein family of Arabidopsis thaliana. Three subfamilies can be distinguished on the basis of structural organization and sequence homology. We investigated the subcellular localization of two members of the largest subfamily, i.e. AtRTNLB2 and AtRTNLB4, using fluorescent protein tags. The results demonstrate for the first time that plant reticulon-like proteins are associated with the ER. Both AtRTNLB proteins are located in the tubular ER but AtRTNLB4 is also found in the lamellar ER cisternae, and in ER tubules in close association with the chloroplasts. Similarity in protein structure and subcellular localization between AtRTNLB2 and mammalian reticulons suggests that they could assume similar basic functions inside the cell.     

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.     

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.     

Chemokine receptor internalization and intracellular trafficking

The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors.     

The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception

Flagellin, the main building block of the bacterial flagellum, acts as a pathogen-associated molecular pattern triggering the innate immune response in animals and plants. In Arabidopsis thaliana, the Leu-rich repeat transmembrane receptor kinase FLAGELLIN SENSITIVE2 (FLS2) is essential for flagellin perception. Here, we demonstrate the specific interaction of the elicitor-active epitope flg22 with the FLS2 protein by chemical cross-linking and immunoprecipitation. The functionality of this receptor was further tested by heterologous expression of the Arabidopsis FLS2 gene in tomato (Lycopersicon esculentum) cells. The perception of flg22 in tomato differs characteristically from that in Arabidopsis. Expression of Arabidopsis FLS2 conferred an additional flg22-perception system on the cells of tomato, which showed all of the properties characteristic of the perception of this elicitor in Arabidopsis. In summary, these results show that FLS2 constitutes the pattern-recognition receptor that determines the specificity of flagellin perception.     

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85-91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.     

Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative beta-arrestin1(319-418) fragment or by expression in beta-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of beta-arrestins.     

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.     

MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment

MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HT_2AR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HT_2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT_2AR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HT_2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT_2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT_2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT_2AR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HT_2AR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HT_2AR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCβ3 to 5-HT_2AR. In addition, we report a negative effect for knocking down MAGI-3 on β-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HT_2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.     

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y₆. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y₆ receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y₆. Interestingly, UDP induced clathrin-dependent P2Y₆ internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y₆ was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y₆ internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y₆ receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y₆ signaling.     

Two new clock proteins, LWD1 and LWD2, regulate Arabidopsis photoperiodic flowering

The “light” signal from the environment sets the circadian clock to regulate multiple physiological processes for optimal rhythmic growth and development. One such process is the control of flowering time by photoperiod perception in plants. In Arabidopsis (Arabidopsis thaliana), the flowering time is determined by the correct interconnection of light input and signal output by the circadian clock. The identification of additional clock proteins will help to better dissect the complex nature of the circadian clock in Arabidopsis. Here, we show LIGHT-REGULATED WD1 (LWD1)/LWD2 as new clock proteins involved in photoperiod control. The lwd1lwd2 double mutant has an early-flowering phenotype, contributed by the significant phase shift of CONSTANS (CO), and, therefore, an increased expression of FLOWERING LOCUS T (FT) before dusk. Under entrainment conditions, the expression phase of oscillator (CIRCADIAN CLOCK ASSOCIATED1 [CCA1], LATE ELONGATED HYPOCOTYL [LHY], TIMING OF CAB EXPRESSION1 [TOC1], and EARLY FLOWERING4 [ELF4]) and output (GIGANTEA, FLAVIN-BINDING, KELCH REPEAT, F-BOX1, CYCLING DOF FACTOR1, CO, and FT) genes in the photoperiod pathway shifts approximately 3 h forward in the lwd1lwd2 double mutant. Both the oscillator (CCA1, LHY, TOC1, and ELF4) and output (COLD, CIRCADIAN RHYTHM, AND RNA BINDING2 and CHLOROPHYLL A/B-BINDING PROTEIN2) genes have a short period length in the lwd1lwd2 double mutant. Our data imply that LWD1/LWD2 proteins function in close proximity to or within the circadian clock for photoperiodic flowering control.     

Probing the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions of specific domains to signaling function

Flagellin sensing2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu receptor that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.     

SUMO-SIM interactions regulate the activity of RGSZ2 proteins

The RGSZ2 gene, a regulator of G protein signaling, has been implicated in cognition, Alzheimer's disease, panic disorder, schizophrenia and several human cancers. This 210 amino acid protein is a GTPase accelerating protein (GAP) on Gαi/o/z subunits, binds to the N terminal of neural nitric oxide synthase (nNOS) negatively regulating the production of nitric oxide, and binds to the histidine triad nucleotide-binding protein 1 at the C terminus of different G protein-coupled receptors (GPCRs). We now describe a novel regulatory mechanism of RGS GAP function through the covalent incorporation of Small Ubiquitin-like MOdifiers (SUMO) into RGSZ2 RGS box (RH) and the SUMO non covalent binding with SUMO-interacting motifs (SIM): one upstream of the RH and a second within this region. The covalent attachment of SUMO does not affect RGSZ2 binding to GPCR-activated GαGTP subunits but abolishes its GAP activity. By contrast, non-covalent binding of SUMO with RH SIM impedes RGSZ2 from interacting with GαGTP subunits. Binding of SUMO to the RGSZ2 SIM that lies outside the RH does not affect GαGTP binding or GAP activity, but it could lead to regulatory interactions with sumoylated proteins. Thus, sumoylation and SUMO-SIM interactions constitute a new regulatory mechanism of RGS GAP function and therefore of GPCR cell signaling as well.     

Interactions with PDZ domain proteins PIST/GOPC and PDZK1 regulate intracellular sorting of the somatostatin receptor subtype 5

By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting.     

The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis

The molecular mechanism governing the response of plants to salinity stress, one of the most significant limiting factors for agriculture worldwide, has just started to be revealed. Here, we report AtSZF1 and AtSZF2, two closely related CCCH-type zinc finger proteins, involved in salt stress responses in Arabidopsis. The expression of AtSZF1 and AtSZF2 is quickly and transiently induced by NaCl treatment. Mutants disrupted in the expression of AtSZF1 or AtSZF2 exhibit increased expression of a group of salt stress-responsive genes in response to high salt. Significantly, the atszf1-1/atszf2-1 double mutant displays more sensitive responses to salt stress than the atszf1-1 or atszf2-1 single mutants and wild-type plants. On the other hand, transgenic plants overexpressing AtSZF1 show reduced induction of salt stress-responsive genes and are more tolerant to salt stress. We also showed that AtSZF1 is localized in the nucleus. Taken together, these results demonstrated that AtSZF1 and AtSZF2 negatively regulate the expression of salt-responsive genes and play important roles in modulating the tolerance of Arabidopsis plants to salt stress.     

PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking

PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.     

E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes

This is an addendum to our recent paper published in The Plant Journal (52:352–61). The major findings were: (1) trichomes on the leaves of gl3-sst sim double mutants developed as large multi-cellular clusters whereas wild type trichomes are composed of single cells; (2) ectopic CYCD3;1 expression in gl3-sst trichomes also resulted in trichome cluster formation; and (3) that GL1 expression is prolonged in the gl3-sst sim trichome clusters. This addendum shows that ectopic CYCD3;1 expression in gl3-sst also enhanced GL1 expression. An analysis of the GL1 promoter found two overlapping potential E2F binding sites in a region of the promoter known to be essential for GL1 function. This finding indicates that GL1 may be directly regulated by the activity of a CYCD3/CDKA complex that phosphorylates E2F-RB bound to the GL1 promoter.Key words: plant cell cycle, endoreduplication, glabra1, plant development