Subcellular Trafficking Research Articles

Profiling the proximal proteome of the activated μ-opioid receptor.

The μ-opioid receptor (μOR) represents an important target of therapeutic and abused drugs. So far, most understanding of μOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet μOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated μOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the μOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new μOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana.

Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germlines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO/PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO/PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana. We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation.

Development and immunological evaluation of an mRNA-based vaccine targeting Naegleria fowleri for the treatment of primary amoebic meningoencephalitis

More than 95% of patients fall victim to primary amoebic meningoencephalitis (PAM), a fatal disease attacking the central nervous system. Naegleria fowleri, a brain-eating microorganism, is PAM's most well-known pathogenic ameboflagellate. Despite the use of antibiotics, the fatality rate continues to rise as no clinical trials have been conducted against this disease. To address this, we mined the UniProt database for pathogenic proteins and selected assumed epitopes to create an mRNA-based vaccine. We identified thirty B-cell and T-cell epitopes for the vaccine candidate. These epitopes, secretion boosters, subcellular trafficking structures, and linkers were used to construct the vaccine candidate. Through predictive modeling and confirmation via the Ramachandran plot (with a quality factor of 92.22), we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: 0.394, instability index: 38.99, antigenicity: 0.8). The theoretical model of immunological simulations indicated favorable responses from both innate and adaptive immune cells, with memory cells expected to remain active for up to 350 days post-vaccination, while the antigen was eliminated from the body within 24 h. Notably, strong interactions were observed between the vaccine construct and TLR-4 (− 11.9 kcal/mol) and TLR-3 (− 18.2 kcal/mol).

Imaging the ER and Endomembrane System in Cereal Endosperm.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the endomembrane system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

VAMP7j: A Splice Variant of Human VAMP7 That Modulates Neurite Outgrowth by Regulating L1CAM Transport to the Plasma Membrane.

The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity.

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature

Introduction: Natriuretic peptide receptor 2 (NPR2 or NPR-B) plays a central role in growth development and bone morphogenesis and therefore loss-of-function variations in NPR2 gene have been reported to cause Acromesomelic Dysplasia, Maroteaux type 1 and short stature. While several hypotheses have been proposed to underlie the pathogenic mechanisms responsible for these conditions, the exact mechanisms, and functional characteristics of many of those variants and their correlations with the clinical manifestations have not been fully established.Methods: In this study, we examined eight NPR2 genetic missense variants (p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg318Gly, p.Arg388Gln, p.Arg495Cys, p.Arg557His, and p.Arg932Cys) Acromesomelic Dysplasia, Maroteaux type 1 and short stature located on diverse domains and broadly classified as variants of uncertain significance. The evaluated variants are either reported in patients with acromesomelic dysplasia in the homozygous state or short stature in the heterozygous state. Our investigation included the evaluation of their expression, subcellular trafficking and localization, N-glycosylation profiles, and cyclic guanosine monophosphate (cGMP) production activity.Results and Discussion: Our results indicate that variants p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg388Gln have defective cellular trafficking, being sequestered within the endoplasmic reticulum (ER), and consequently impaired cGMP production ability. Conversely, variants p.Arg318Gly, p.Arg495Cys, and p.Arg557His seem to display a non-statistically significant behavior that is slightly comparable to WT-NPR2. On the other hand, p.Arg932Cys which is located within the guanylyl cyclase active site displayed normal cellular trafficking profile albeit with defective cGMP. Collectively, our data highlights the genotype-phenotype relationship that might be responsible for the milder symptoms observed in short stature compared to acromesomelic dysplasia. This study enhances our understanding of the functional consequences of several NPR2 variants, shedding light on their mechanisms and roles in related genetic disorders which might also help in their pathogenicity re-classification.

Promoter and domain structures regulate FLA12 function during Arabidopsis secondary wall development.

Fasciclin-like arabinogalactan-proteins (FLAs) are a family of multi-domain glycoproteins present at the cell surface and walls of plants. Arabidopsis thaliana FLA12 and homologs in cotton, Populus, and flax have been shown to play important functions regulating secondary cell wall (SCW) development. FLA12 has been shown to have distinct roles from the closely related FLA11 that also functions during SCW development. The promoter and domain features of FLA12 that regulate functional specificity have not been well characterized. In this study, promoter swap experiments of FLA11 and FLA12 were investigated. Mutation of proposed functional regions within FLA12 were used to investigate the role of post-translational modifications on sub-cellular location and trafficking. Domain swap experiments between FLA11 and FLA12 were performed to identify regions of functional specificity. Promote swap experiments showed that FLA12 is differentially expressed in both stem and rosette leaves compared to FLA11. Post-translational modifications, in particular addition of the glycosylphosphatidylinositol-anchor (GPI-anchor), were shown to be important for FLA12 location at the plasma membrane (PM)/cell wall interface. Domain swap experiments between FLA11 and FLA12 showed that the C-terminal arabinogalactan (AG) glycan motif acts as a key regulatory region differentiating FLA12 functions from FLA11. Understanding of FLA12 promoter and functional domains has provided new insights into the regulation of SCW development and functional specificity of FLAs for plant growth and development.

ATG9B is a tissue-specific homotrimeric lipid scramblase that can compensate for ATG9A

ABSTRACT Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophagy-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A vertebrates encode ATG9B, a poorly characterized paralog expressed in a subset of tissues. Herein, we characterize the structure of human ATG9B revealing the conserved homotrimeric quaternary structure and explore the conformational dynamics of the protein. Consistent with the experimental structure and computational chemistry, we establish that ATG9B is a functional lipid scramblase. We show that ATG9B can compensate for the absence of ATG9A in starvation-induced autophagy displaying similar subcellular trafficking and steady-state localization. Finally, we demonstrate that ATG9B can form a heteromeric complex with ATG2A. By establishing the molecular structure and function of ATG9B, our results inform the exploration of niche roles for autophagy machinery in more complex eukaryotes and reveal insights relevant across species. Abbreviation: ATG: autophagy related; CHS: cholesteryl hemisuccinate; cryo-EM: single-particle cryogenic electron microscopy; CTF: contrast transfer function: CTH: C- terminal α helix; FSC: fourier shell correlation; HDIR: HORMA domain interacting region; LMNG: lauryl maltose neopentyl glycol; MD: molecular dynamics simulations; MSA: multiple sequence alignment; NBD-PE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl ammonium salt); POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RBG: repeating beta groove domain; RMSD: root mean square deviation; SEC: size-exclusion chromatography; TMH: transmembrane helix

Subcellular trafficking and transcytosis efficacy of different receptor types for therapeutic antibody delivery at the blood‒brain barrier

Here, we report an experimental setup to benchmark different receptors for targeted therapeutic antibody delivery at the blood–brain barrier. We used brain capillary endothelial-like cells derived from induced pluripotent stem cells (hiPSC-BECs) as a model system and compared them to colon epithelial Caco-2 cells. This approach helped to identify favourable receptors for transport into the cell layer itself or for directing transport for transcytosis across the cell layer. The sorting receptors transferrin receptor and sortilin were shown to be efficient as antibody cargo receptors for intracellular delivery to the cell layer. In contrast, the cell surface receptors CD133 and podocalyxin were identified as static and inefficient receptors for delivering cargo antibodies. Similar to in vivo studies, the hiPSC-BECs maintained detectable transcytotic transport via transferrin receptor, while transcytosis was restricted using sortilin as a cargo receptor. Based on these findings, we propose the application of sortilin as a cargo receptor for delivering therapeutic antibodies into the brain microvascular endothelium.Graphical

Cellular roadmaps of viroid infection.

Viroids are single-stranded circular noncoding RNAs that infect plants. According to the International Committee on Taxonomy of Viruses, there are 44 viroids known to date. Notably, more than 20 000 distinct viroid-like RNA sequences have recently been identified in existing sequencing datasets, suggesting an unprecedented complexity in biological roles of viroids and viroid-like RNAs. Interestingly, a human pathogen, hepatitis delta virus (HDV), also replicates via a rolling circle mechanism like viroids. Therefore, knowledge of viroid infection is informative for research on HDV and other viroid-like RNAs reported from various organisms. Here, we summarize recent advancements in understanding viroid shuttling among subcellular compartments for completing replication cycles, emphasizing regulatory roles of RNA motifs and structural dynamics in diverse biological processes. We also compare the knowledge of viroid intracellular trafficking with known pathways governing cellular RNA movement in cells. Future investigations on regulatory RNA structures and cognate factors in regulating viroid subcellular trafficking and replication will likely provide new insights into RNA structure-function relationships and facilitate the development of strategies controlling RNA localization and function in cells.

Low throughput screening in neuroscience: using light to study central synapses one at a time.

Neurophotonic approaches have fostered substantial progress in our understanding of the brain by providing an assortment of means to either monitor or manipulate neural processes. Among these approaches, the development of two-photon uncaging provides a useful and flexible approach to manipulate the activity of individual synapses. In this short piece, we explore how this technique has emerged at the intersection of chemistry, optics, and electrophysiology to enable spatially and temporally precise photoactivation for studying functional aspects of synaptic transmission and dendritic integration. We discuss advantages and limitations of this approach, focusing on our efforts to study several functional aspects of glutamate receptors using uncaging of glutamate. Among other advancements, this approach has contributed to further our understanding of the subcellular regulation, trafficking, and biophysical features of glutamate receptors (e.g., desensitization and silent synapse regulation), the dynamics of spine calcium, and the integrative features of dendrites, and how these functions are altered by several forms of plasticity.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

Cytoplasmic FKBPs are involved in molting and metamorphosis through regulating the nuclear localization of EcR.

Molting and metamorphosis are important physiological processes in insects that are tightly controlled by ecdysone receptor (EcR) through the 20-hydroxyecdysone (20E) signaling pathway. EcR is a steroid nuclear receptor (SR). Several FK506-binding proteins (FKBPs) have been identified from the mammal SR complex, and are thought to be involved in the subcellular trafficking of SR. However, their roles in insects are poorly understood. To explore whether FKBPs are involved in insect molting or metamorphosis, we injected an FKBP inhibitor (FK506) into a lepidopteran insect, Spodoptera litura, and found that molting was inhibited in 61.11% of the larvae, and that the time for larvae to pupate was significantly extended. A total of 10 FKBP genes were identified from the genome of S.litura and were clustered into 2 distinct groups, according to their subcellular localization, with FKBP13 and FKBP14 belonging to the endoplasmic reticulum (ER) group and with the other members belonging to the cytoplasmic (Cy) group. All the CyFKBPs were significantly upregulated in the prepupal or pupal stages, with the opposite being observed for the ER group members. FK506 completely blocked the transfer of EcR to the nucleus under 20E induction, and significantly downregulated the transcriptional expression of many 20E signaling genes. A similar phenomenon was observed after RNA interference of 2 CyFKBPs (FKBP45 and FKBP12b), but not for FKBP13. Taken together, our data indicate that the cytoplasmic FKBPs, especially FKBP45 and FKBP12b, mediate the nuclear localization of EcR, thereby regulating the 20E signaling and ultimately affecting molting and metamorphosis in insects.

TRAPPC6B biallelic variants cause a neurodevelopmental disorder with TRAPP II and trafficking disruptions.

Highly conserved transport protein particle (TRAPP) complexes regulate subcellular trafficking pathways. Accurate protein trafficking has been increasingly recognized to be critically important for normal development, particularly in the nervous system. Variants in most TRAPP complex subunits have been found to lead to neurodevelopmental disorders with diverse but overlapping phenotypes. We expand on limited prior reports on TRAPPC6B with detailed clinical and neuroradiologic assessments, and studies on mechanisms of disease, and new types of variants. We describe 29 additional patients from 18 independent families with biallelic variants in TRAPPC6B. We identified seven homozygous nonsense (n = 12 patients) and eight canonical splice-site variants (n = 17 patients). In addition, we identified one patient with compound heterozygous splice-site/missense variants with a milder phenotype and one patient with homozygous missense variants. Patients displayed non-progressive microcephaly, global developmental delay/intellectual disability, epilepsy and absent expressive language. Movement disorders including stereotypies, spasticity and dystonia were also observed. Brain imaging revealed reductions in cortex, cerebellum and corpus callosum size with frequent white matter hyperintensity. Volumetric measurements indicated globally diminished volume rather than specific regional losses. We identified a reduced rate of trafficking into the Golgi apparatus and Golgi fragmentation in patient-derived fibroblasts that was rescued by wild-type TRAPPC6B. Molecular studies revealed a weakened interaction between mutant TRAPPC6B (c.454C>T, p.Q152*) and its TRAPP binding partner TRAPPC3. Patient-derived fibroblasts from the TRAPPC6B (c.454C>T, p.Q152*) variant displayed reduced levels of TRAPPC6B as well as other TRAPP II complex-specific members (TRAPPC9 and TRAPPC10). Interestingly, the levels of the TRAPPC6B homologue TRAPPC6A were found to be elevated. Moreover, co-immunoprecipitation experiments showed that TRAPPC6A co-precipitates equally with TRAPP II and TRAPP III, while TRAPPC6B co-precipitates significantly more with TRAPP II, suggesting enrichment of the protein in the TRAPP II complex. This implies that variants in TRAPPC6B may preferentially affect TRAPP II functions compared to TRAPP III functions. Finally, we assessed phenotypes in a Drosophila TRAPPC6B-deficiency model. Neuronal TRAPPC6B knockdown impaired locomotion and led to wing posture defects, supporting a role for TRAPPC6B in neuromotor function. Our findings confirm the association of damaging biallelic TRAPPC6B variants with microcephaly, intellectual disability, language impairments, and epilepsy. A subset of patients also exhibited dystonia and/or spasticity with impaired ambulation. These features overlap with disorders arising from pathogenic variants in other TRAPP subunits, particularly components of the TRAPP II complex. These findings suggest that TRAPPC6B is essential for brain development and function, and TRAPP II complex activity may be particularly relevant for mediating this function.

Pallidin function in Drosophila surface glia regulates sleep and is dependent on amino acid availability

The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. We show here that downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and we identify a role for Pallidin in the subcellular trafficking of JhI-21. Finally, we provide evidence that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation.

Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.

Alternative splicing regulates adaptor protein binding, trafficking, and activity of the Vps10p domain receptor SorCS2 in neuronal development

The Vps10p domain receptor SorCS2 is crucial for the development and function of the nervous system and essential for brain-derived neurotrophic factor (BDNF)-induced changes in neuronal morphology and plasticity. SorCS2 regulates the subcellular trafficking of the BDNF signaling receptor TrkB as well as selected neurotransmitter receptors in a manner that is dependent on the SorCS2 intracellular domain (ICD). However, the cellular machinery and adaptor protein (AP) interactions that regulate receptor trafficking via the SorCS2 ICD are unknown. We here identify four splice variants of human SorCS2 differing in the insertion of an acidic cluster motif and/or a serine residue within the ICD. We show that each variant undergoes posttranslational proteolytic processing into a one- or two-chain receptor, giving rise to eight protein isoforms, the expression of which differs between neuronal and nonneuronal tissues and is affected by cellular stressors. We found that the only variants without the serine were able to rescue BDNF-induced branching of SorCS2 knockout hippocampal neurons, while variants without the acidic cluster showed increased interactions with clathrin-associated APs AP-1, AP-2, and AP-3. Using yeast two-hybrid screens, we further discovered that all variants bound dynein light chain Tctex-type 3; however, only variants with an acidic cluster motif bound kinesin light chain 1. Accordingly, splice variants showed markedly different trafficking properties and localized to different subcellular compartments. Taken together, our findings demonstrate the existence of eight functional SorCS2 isoforms with differential capacity for interactions with cytosolic ligands dynein light chain Tctex-type 3 and kinesin light chain 1, which potentially allows cell-type specific SorCS2 trafficking and BDNF signaling.

Interaction of Human OATP1B1 with PDZK1 Is Required for Its Trafficking to the Hepatocyte Plasma Membrane.

Uptake of xenobiotics by hepatocytes is mediated by specific proteins, including organic anion transporting polypeptides (OATPs), residing on the basolateral (sinusoidal) plasma membrane. Many of the OATPs have PDZ consensus binding sites, determined by their C-terminal 4 amino acids, while others do not. Mouse and rat OATP1A1 are associated with PDZK1, which is necessary for their trafficking to the plasma membrane. humanOATP1B1 (hOATP1B1) is a major drug transporter in human liver. Although localized to the plasma membrane, it was thought to lack a PDZ consensus motif, suggesting that the trafficking paradigm for murine OATPs is not applicable to human liver. The aim of the present study was to determine whether hOATP1B1 is a ligand for hPDZK1. hOATP1B1 immunoprecipitates with hPDZK1 following co-expression in 293T cells as well as in normal human liver. Co-expression with each of the 4 PDZ domains revealed interaction with domain 1 only. A truncated version of hOATP1B1 that lacks its terminal 4 amino acid PDZ binding motif as well as hOATP1B3, which does not contain a PDZ binding consensus motif, failed to interact with hPDZK1. Immunofluorescence microscopy of hOATP1B1 in stably transfected HeLa cells that endogenously express hPDZK1 showed that it distributes predominantly along the plasma membrane whereas hOATP1B1 lacking its terminal 4 amino acids distributes primarily intracellularly with little plasma membrane localization. Similar to findings in rats and mice, human OATP1B1 is a ligand for PDZK1 and requires interaction with PDZK1 for optimal trafficking to the hepatocyte plasma membrane. SIGNIFICANCE: Previous studies suggested that OATP1B1, a major xenobiotic transporter in human liver, does not have a PDZ binding consensus motif and does not follow the paradigm for subcellular trafficking and function that was established for OATP1A1 in murine liver. We now demonstrated that OATP1B1 but not OATP1B3 has a PDZ binding consensus motif that mediates binding to PDZK1 and is required for its trafficking to the plasma membrane. Such interaction could be an important previously unrecognized modulator of transport function.

Ubiquitination of the GluA1 Subunit of AMPA Receptors Is Required for Synaptic Plasticity, Memory, and Cognitive Flexibility.

Activity-dependent changes in the number of AMPA-type glutamate receptors (AMPARs) at the synapse underpin the expression of LTP and LTD, cellular correlates of learning and memory. Post-translational ubiquitination has emerged as a key regulator of the trafficking and surface expression of AMPARs, with ubiquitination of the GluA1 subunit at Lys-868 controlling the post-endocytic sorting of the receptors into the late endosome for degradation, thereby regulating their stability at synapses. However, the physiological significance of GluA1 ubiquitination remains unknown. In this study, we generated mice with a knock-in mutation in the major GluA1 ubiquitination site (K868R) to investigate the role of GluA1 ubiquitination in synaptic plasticity, learning, and memory. Our results reveal that these male mice have normal basal synaptic transmission but exhibit enhanced LTP and deficits in LTD. They also display deficits in short-term spatial memory and cognitive flexibility. These findings underscore the critical roles of GluA1 ubiquitination in bidirectional synaptic plasticity and cognition in male mice.SIGNIFICANCE STATEMENT Subcellular targeting and membrane trafficking determine the precise number of AMPA-type glutamate receptors at synapses, processes that are essential for synaptic plasticity, learning, and memory. Post-translational ubiquitination of the GluA1 subunit marks AMPARs for degradation, but its functional role in vivo remains unknown. Here we demonstrate that the GluA1 ubiquitin-deficient mice exhibit an altered threshold for synaptic plasticity accompanied by deficits in short-term memory and cognitive flexibility. Our findings suggest that activity-dependent ubiquitination of GluA1 fine-tunes the optimal number of synaptic AMPARs required for bidirectional synaptic plasticity and cognition in male mice. Given that increases in amyloid-β cause excessive ubiquitination of GluA1, inhibiting that GluA1 ubiquitination may have the potential to ameliorate amyloid-β-induced synaptic depression in Alzheimer's disease.