Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson's disease.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Parkinson's disease risk genes act in glia to control neuronal α-synuclein toxicity.

Idiopathic Parkinson's disease is the second most common neurodegenerative disease and is estimated to be approximately 30% heritable. Genome wide association studies have revealed numerous loci associated with risk of development of Parkinson's disease. The majority of genes identified in these studies are expressed in glia at either similar or greater levels than their expression in neurons, suggesting that glia may play a role in Parkinson's disease pathogenesis. The role of individual glial risk genes in Parkinson's disease development or progression is unknown, however. We hypothesized that some Parkinson's disease risk genes exert their effects through glia. We developed a Drosophila model of α-synucleinopathy in which we can independently manipulate gene expression in neurons and glia. Human wild type α-synuclein is expressed in all neurons, and these flies develop the hallmarks of Parkinson's disease, including motor impairment, death of dopaminergic and other neurons, and α-synuclein aggregation. In these flies, we performed a candidate genetic screen, using RNAi to knockdown 14 well-validated Parkinson's disease risk genes in glia and measuring the effect on locomotion in order to identify glial modifiers of the α-synuclein phenotype. We identified 4 modifiers: aux, Lrrk, Ric, and Vps13, orthologs of the human genes GAK, LRRK2, RIT2, and VPS13C, respectively. Knockdown of each gene exacerbated neurodegeneration as measured by total and dopaminergic neuron loss. Knockdown of each modifier also increased α-synuclein oligomerization. These results suggest that some Parkinson's disease risk genes exert their effects in glia and that glia can influence neuronal α-synuclein proteostasis in a non-cell-autonomous fashion. Further, this study provides proof of concept that our novel Drosophila α-synucleinopathy model can be used to study glial modifier genes, paving the way for future large unbiased screens to identify novel glial risk factors that contribute to PD risk and progression.

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components.

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Identification of a molecular marker associated with lignotuber in Eucalyptus ssp.

About 95% of Eucalyptus species present an organ known as a lignotuber, a basal woody swelling that holds a large number of dormant buds in a protected position along with carbohydrates and other nutrients. The importance of this trait in Eucalyptus species relates to its regenerative capacity, particularly in the context of coppicing practices and survival in regions of high abiotic stress, especially fire. In this study, we identified and characterized a genomic region associated with the lignotuber trait in commercially important Eucalyptus species by developing a polymorphic marker that co-segregates with lignotuber presence. The marker was then converted into a SCAR (Sequence Characterized Amplified Region) marker, validated in four other Eucalyptus species and hybrids and analyzed in silico. Our investigation presents a marker (ELig) that is effective in identifying individuals with lignotuber. In silico and Southern blot analyses show that the marker is present in a single copy region and is related to auxilin/cyclin-G associated kinase, containing a DnaJ domain. The ELig marker is an important tool that can be used to manage crosses in Eucalyptus breeding programs and inform studies involving lignotuber development and genetics.

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

Mapping and identification of CsUp, a gene encoding an Auxilin-like protein, as a putative candidate gene for the upward-pedicel mutation (up) in cucumber.

BACKGROUND: Pedicel orientation can affect the female flower orientation and seed yield in cucumber. A spontaneous mutant possessing upward growth of pedicels was identified in the wild type inbred strain 9930 and named upward-pedicel (up). The morphological and genetic analyses of up were performed in this study. In order to clone the up gene, 933 F2 individuals and 524 BC1 individuals derived from C-8-6 (WT) and up were used for map-based cloning. RESULTS: up was mapped to a 35.2 kb physical interval on chromosome 1, which contains three predicted genes. Sequencing analysis revealed that a 5-bp deletion was found in the second exon of Csa1G535800, and it led to a frameshift mutation resulting in a premature stop codon. The candidate gene of CsUp (Csa1G535800) was further confirmed via genomic and cDNA sequencing in biparental and natural cucumber populations. Sequencing data showed that a 4-bp deletion was found in the sixth exon of Csa1G535800 in CGN19839, another inbred line, and there was also a mutation of an amino acid in Csa1G535800 that could contribute to the upward growth of pedicels in CGN19839. Moreover, it was found that Csa1G535800 exhibited strong expression in the pedicel of WT, suggesting its important role in development of pedicel orientation. Thus, Csa1G535800 was considered to be the candidate gene of CsUp. CONCLUSIONS: CsUp encodes an Auxilin-like protein and controls pedicel orientation in cucumber. The identification of CsUp may help us to understand the mechanism of pedicel orientation development and allow for investigation of novel functions of Auxilin-like proteins in cucumber.

Cargo regulates clathrin-coated pit invagination via clathrin light chain phosphorylation.

Clathrin light chains (CLCs) control selective uptake of a range of G protein-coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.

LRRK2 phosphorylation of auxilin mediates synaptic defects in dopaminergic neurons from patients with Parkinson's disease.

Recently identified Parkinson's disease (PD) genes involved in synaptic vesicle endocytosis, such as DNAJC6 (auxilin), have further implicated synaptic dysfunction in PD pathogenesis. However, how synaptic dysfunction contributes to the vulnerability of human dopaminergic neurons has not been previously explored. Here, we demonstrate that commonly mutated, PD-linked leucine-rich repeat kinase 2 (LRRK2) mediates the phosphorylation of auxilin in its clathrin-binding domain at Ser627. Kinase activity-dependent LRRK2 phosphorylation of auxilin led to differential clathrin binding, resulting in disrupted synaptic vesicle endocytosis and decreased synaptic vesicle density in LRRK2 patient-derived dopaminergic neurons. Moreover, impaired synaptic vesicle endocytosis contributed to the accumulation of oxidized dopamine that in turn mediated pathogenic effects such as decreased glucocerebrosidase activity and increased α-synuclein in mutant LRRK2 neurons. Importantly, these pathogenic phenotypes were partially attenuated by restoring auxilin function in mutant LRRK2 dopaminergic neurons. Together, this work suggests that mutant LRRK2 disrupts synaptic vesicle endocytosis, leading to altered dopamine metabolism and dopamine-mediated toxic effects in patient-derived dopaminergic neurons.

Auxilin facilitates membrane traffic in the early secretory pathway.

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis.

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1's function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1's regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis.

Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE3-1].

Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion-specific requirements for the propagation of the [URE3] prion variant [URE3-1], we screened 12 yeast cytosolic J-proteins, and here we report a novel role for the J-protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle-mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE3-1] is specifically dependent upon Swa2, but not on any of the 11 other J-proteins. Further, we show that [URE3-1] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2-clathrin binding. Because the J-domain of Swa2 can be replaced with the J-domains of other proteins, our data strongly suggest that prion-chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation.

A deleterious mutation in DNAJC6 encoding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkinsonism.

Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of parkinsonism.

MiR-3120 is a mirror microRNA that targets heat shock cognate protein 70 and auxilin messenger RNAs and regulates clathrin vesicle uncoating.

We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin.

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling.

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis.

Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.

Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis.

Chloroplasts move toward weak light (accumulation response) and away from strong light (avoidance response). The fast and accurate movement of chloroplasts in response to ambient light conditions is essential for efficient photosynthesis and photodamage prevention in chloroplasts. Here, we report that two Arabidopsis mutants, weak chloroplast movement under blue light 1 (web1) and web2, are defective in both the avoidance and the accumulation responses. Map-based cloning revealed that both genes encode coiled-coil proteins and that WEB2 is identical to the plastid movement impaired 2 (PMI2) gene. The velocities of chloroplast movement in web1 and pmi2 were approximately threefold lower than that in the wild type. Defects in the avoidance response of web1 and pmi2 were suppressed by mutation of the J-domain protein required for chloroplast accumulation response 1 (JAC1) gene, which is essential for the accumulation response; these results indicate that WEB1 and PMI2 play a role in suppressing JAC1 under strong light conditions. A yeast two-hybrid analysis and a nuclear recruitment assay identified a physical interaction between WEB1 and PMI2, and transient expression analysis of CFP-WEB1 and YFP-PMI2 revealed that they colocalized in the cytosol. Bimolecular fluorescence complementation analysis confirmed the interaction of these proteins in the cytosol. Blue light-induced changes in short chloroplast actin filaments (cp-actin filaments) were impaired in both web1 and pmi2. Our findings suggest that a cytosolic WEB1-PMI2 complex maintains the velocity of chloroplast photorelocation movement via cp-actin filament regulation.

Crystallographic and functional analyses of J-domain of JAC1 essential for chloroplast photorelocation movement in Arabidopsis thaliana.

An auxilin-like J-domain-containing protein, JAC1, is necessary for chloroplast movement in Arabidopsis thaliana, to capture photosynthetic light efficiently under weak light conditions. Here, we performed crystallographic and functional analyses of the J-domain of JAC1. The crystal structure of the J-domain is quite similar to that of bovine auxilin, and possesses a similar positively charged surface, which probably forms the interface with the Hsp70 chaperone. The mutation of the highly conserved HPD motif of the JAC1 J-domain abrogated the chloroplast photorelocation response. These results suggest that the requirement of JAC1 in chloroplast photorelocation movement is attributable to the J-domain's cochaperone activity.