MTB-3, a Microtubule Plus-End Tracking Protein (+TIP) of Neurospora crassa

The microtubule (MT) “plus end” constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called “MT plus-end tracking proteins” (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

dRecQ4 Is Required for DNA Synthesis and Essential for Cell Proliferation in Drosophila

Background

The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging.

Methodology/Principal Findings

To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4.

Conclusions/Significance

Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.     

HIF3A DNA Methylation Is Associated with Childhood Obesity and ALT

Gene polymorphisms associated so far with body mass index (BMI) can explain only 1.18–1.45% of observed variation in BMI. Recent studies suggest that epigenetic modifications, especially DNA methylation, could contribute to explain part of the missing heritability, and two epigenetic genome-wide analysis studies (EWAS) have reported that Hypoxia Inducible Factor 3 Alpha Subunit (HIF3A) methylation was associated with BMI or BMI change. We therefore assessed whether the HIF3A methylation is associated with obesity and other obesity-related phenotypes in Chinese children. The subjects included 110 severe obese cases aged 7–17y and 110 normal-weight controls matched by age and gender for measurement of blood DNA methylation levels at the HIF3A gene locus using the Sequenom’s MassARRAY system. We observed significantly higher methylation levels in obese children than in controls at positions 46801642 and 46801699 in HIF3A gene (P<0.05), and found positive associations between methylation and alanine aminotransferase (ALT) levels adjusted by gender, age and BMI at the position 46801699 (r = 0.226, P = 0.007). These results suggest that HIF3A DNA methylation is associated with childhood obesity, and has a BMI-independent association with ALT. The results provide evidence for identifying epigenetic factors of elivated ALT and may be useful for risk assessment and personalized medicine of liver diseases such as non-alcoholic fatty liver disease (NAFLD).     

Epigenetic Control of a Transposon-Inactivated Gene in Neurospora Is Dependent on DNA Methylation

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5'' noncoding sequences was found in previous work. We report that reversion to Am(+) depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am(+) conidia, whether the cultures were derived from Am(-) or Am(+) isolates. Reversion to Am(+) did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am(+). The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSamα and β, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.     

The Adaptor Protein Paxillin Is Essential for Normal Development in the Mouse and Is a Critical Transducer of Fibronectin Signaling

The integrin family of cell adhesion receptors are important for a diverse set of biological responses during development. Although many integrins have been shown to engage a similar set of cytoplasmic effector proteins in vitro, the importance of these proteins in the biological events mediated by different integrin receptors and ligands is uncertain. We have examined the role of one of the best-characterized integrin effectors, the focal adhesion protein paxillin, by disruption of the paxillin gene in mice. Paxillin was found to be critically involved in regulating the development of mesodermally derived structures such as heart and somites. The phenotype of the paxillin(-/-) mice closely resembles that of fibronectin(-/-) mice, suggesting that paxillin is a critical transducer of signals from fibronectin receptors during early development. Paxillin was also found to play a critical role in fibronectin receptor biology ex vivo since cultured paxillin-null fibroblasts display abnormal focal adhesions, reduced cell migration, inefficient localization of focal adhesion kinase (FAK), and reduced fibronectin-induced phosphorylation of FAK, Cas, and mitogen-activated protein kinase. In addition, we found that paxillin-null fibroblasts show some defects in the cortical cytoskeleton and cell spreading on fibronectin, raising the possibility that paxillin could play a role in structures distinct from focal adhesions. Thus, paxillin and fibronectin regulate some common embryonic developmental events, possibly due to paxillin modulation of fibronectin-regulated focal adhesion dynamics and organization of the membrane cytoskeletal structures that regulate cell migration and spreading.     

The Adaptor Protein Grb2 Is Not Essential for the Establishment of the Glomerular Filtration Barrier

The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.     

The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3_K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 _K499A strain. The ATPase activity of Mcm2-7_3K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.     

The Yeast Adaptor Protein Complex,AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 μ chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Δ vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Δ mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3–dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.The formation of vesicles for transport between membrane-bound organelles requires assembly of coat proteins that are recruited from the cytosol. These proteins direct the sequestration and concentration of cargo as well as invagination of the membrane. One of the best studied classes of coats involved in vesicle budding is comprised of clathrin and its adaptor proteins (APs)¹, AP-1 and AP-2 (Schmid, 1997). In clathrin-mediated vesicle transport the AP complexes play the dual role of cargo selection and recruitment of clathrin to the membrane. These adaptors are heterotetramers containing two large chains (adaptins, α or γ and β), one medium chain (μ), and one small chain (σ). AP-1 (γ, β1, μ1, and σ1) functions in sorting at the TGN, whereas AP-2 (α, β2, μ2, and σ2) is involved in receptor capture at the PM during endocytosis.Although there is a great deal of evidence supporting the involvement of adaptors in clathrin-mediated vesicle budding, recent studies in animal cells have led to the discovery of a novel adaptor-like complex, AP-3, that seems to function independently of clathrin (Newman et al., 1995; Simpson et al., 1996). AP-3 has identical subunit architecture to AP-1 and AP-2, with two adaptin-like subunits (δ and β3), a medium chain (μ3), and a small chain (σ3) (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a , b ). AP-3 antibodies label a perinuclear region, perhaps the TGN, and punctate structures extending to the cell periphery, which may be endosomal compartments (Simpson et al., 1996, 1997; Dell''Angelica et al., 1997a ). However, the mammalian AP-3 complex does not colocalize with clathrin or AP-1 and AP-2 adaptors in cells and it does not copurify with brain clathrin-coated vesicles (Newman et al., 1995; Simpson et al., 1996, 1997; Dell''Angelica et al., 1997b ). Clues to the function of AP-3 have come from the discovery that the garnet gene of Drosophila encodes a protein closely related to δ adaptin (Ooi et al., 1997; Simpson et al., 1997). Mutations in garnet cause decreased pigmentation of the eyes and other tissues and a reduced number of pigment granules, which may be lysosome-like organelles (Ooi et al., 1997; Simpson et al., 1997). Thus, AP-3 is proposed to function in clathrin-independent transport between the TGN, endosomes and/or lysosomes, although its exact sorting function is still not known.Over the last several years, yeast homologues of the mammalian adaptor subunits have been identified, allowing for the examination of specific functions of these proteins in a genetically tractable organism. Genes encoding subunits sufficient for at least three complete AP complexes have been identified by sequence homology (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995) or by function (Panek et al., 1997). APL1-APL6 encode large chain/ adaptin-related subunits, APM1-APM4 encode μ-like chains, and APS1-APS3 are genes for σ-related proteins. Apl2p (β), Apl4p (γ), Apm1p (μ1), and Aps1p (σ1) are thought to be subunits of an AP-1–like complex that functions with clathrin at the late Golgi/TGN (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). Mutations in the yeast AP-1 genes enhance the growth and the α-factor processing defects of a temperature sensitive (ts) allele of the clathrin heavy chain gene (Phan et al., 1994; Rad et al., 1995; Stepp et al., 1995; Payne, G., personal communication). The latter phenotype is a hallmark of clathrin-deficient yeast, in which late Golgi/ TGN proteins, such as the α-factor processing enzymes Kex2p and dipeptidyl amino peptidase-A (DPAP)-A, are not retained in the late Golgi but escape to the cell surface (Seeger and Payne, 1992b ). To date, no yeast adaptor subunit has been shown to be important for endocytosis, although Apl3p, Apm4p, and Aps2p are most homologous to mammalian AP-2 α, μ2 and σ2, respectively.Recently, a yeast adaptor related to AP-3 of animal cells was described (Panek et al., 1997). It is comprised of Apl5p, Apl6p, Apm3p, and Aps3p, which show preferential homology to mammalian δ, β3, μ3, and σ3, respectively. Mutations in each of these subunits were isolated by their ability to suppress the lethality resulting from loss of function of PM casein kinase 1 encoded by a gene pair, YCK1 and YCK2. Yck activity was found to be required for constitutive endocytosis of the a-factor receptor (Ste3p), and AP-3 subunit mutations partially rescued this internalization defect (Panek et al., 1997). However, the AP complex itself is not necessary for endocytosis, nor is it required for sorting of carboxypeptidase Y (CPY) or retention of late Golgi proteins. Furthermore, unlike disruption of the yeast AP-1 complex, loss of AP-3 function causes no synthetic phenotype in combination with chc1 mutations, suggesting it may function independently of clathrin. Although these data indicated that Apl5p, Apl6p, Apm3p, and Aps3p comprise an AP-3-like adaptor, its precise sorting role was still not known.In this report we describe a genetic approach to determine the function of the yeast AP-3 complex. A colony sectoring screen was performed to identify genes that are essential in the absence of Apm3p, the yeast AP-3 μ chain. Such synthetic lethal screens can be used to identify functional homologues, genes whose proteins function in intersecting or parallel pathways, and genes whose proteins physically interact (Bender and Pringle, 1991). We have cloned the gene for the apm three synthetic lethal mutant, mts1-1, and found it encodes Vps45p, a protein involved in vacuolar protein sorting (Vps; Cowles et al., 1994; Piper et al., 1994). The Vps pathway is defined by >40 complementation groups whose proteins are required for the transport of a number of soluble and membrane-bound proteins, including CPY, protease A (PrA), and carboxypeptidase S (CPS) from the late Golgi/TGN to the vacuole (Stack et al., 1995; Cowles et al., 1997). This pathway is also essential for proper assembly of the vacuolar ATPase (Raymond et al., 1992). However, the type II vacuolar membrane protein alkaline phosphatase (ALP) follows an alternate intracellular pathway to the vacuole (Raymond et al., 1992; Nothwehr et al., 1995; Cowles et al., 1997; Piper et al., 1997). Few vps mutants prevent localization of ALP to the vacuolar membrane and its arrival at the vacuole is not dependent upon transport through the cell surface. The requirement for Apm3p in the absence of Vps45p suggested the possibility that at least one of these routes to the vacuole must be functional for survival and led us to examine ALP sorting in the AP-3 mutants. We show here that yeast AP-3 is essential for the transport of ALP via the alternative pathway to the vacuole.     

BTB Protein KLHL12 targets the dopamine D4 receptor for ubiquitination by a Cul3-based E3 ligase

Dopamine receptors belong to the superfamily of G-protein-coupled receptors and are subdivided into D1-type (D1 and D5) and D2-type (D2, D3, and D4) receptors. The D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated with, among other conditions, attention deficit hyperactivity disorder. Here, we demonstrate that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor through its Kelch domain. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. In addition, we show that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity toward the D4 receptor.