The monomeric clathrin assembly protein, AP180, regulates contractile vacuole size in Dictyostelium discoideum

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.     

Compromise of clathrin function and membrane association by clathrin light chain deletion

While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA– mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium . In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA– mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA– mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo .     

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.     

A single β adaptin contributes to AP1 and AP2 complexes and clathrin function in Dictyostelium

The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a β subunit. We identified a single β subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the β subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single β subunit to the stability and function of AP1 and AP2 in a simple eukaryote.     

Characterization of the clathrin heavy chain from Dictyostelium discoideum.

We report the cloning and analysis of a clathrin heavy-chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the clathrin heavy chain encoded a protein of 1,694 amino acids with a molecular mass of 193,618 daltons. Comparison of the amino acid sequence with the rat and with the yeast sequence showed that the highly conserved protein was more similar to the mammalian clathrin heavy chain (57% identity) than to the yeast heavy chain (45% identity). The mRNA for the clathrin heavy chain was regulated during development. mRNA levels were highest during vegetative growth and declined as the cells progressed through the 24-hr developmental cycle. The concentration of clathrin heavy-chain protein was the same in cells grown in liquid media (high rates of pinocytosis) as in cells grown with bacteria (low rates of pinocytosis), which suggests that regulation of pinocytosis in these cells is not achieved by altering the concentration of clathrin.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells

Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo.     

Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.     

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a- factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild- type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.     

A 220 kDa polypeptide, immunolocalized to epithelial tight junctions, is associated with brain clathrin preparations

Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Characterization of Clathrin-Coated Vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas rein-hardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter±SD of 95±17 nm, n=300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.     

Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.     

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.     

Clathrin is spindle-associated but not essential for mitosis

Background

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

Methodology/Principal Findings

Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

Conclusions/Significance

We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.     

A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the alpha-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.     

Purification and characterization of clathrin-coated vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.