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PubMed1996

Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes.

Clathrin-coated vesicles were isolated from soybean (Glycine max L.) cells in suspension culture and their purity was assessed using SDS-PAGE, peptide sequencing and electron microscopy. Antibodies raised to these coated vesicles were used to immunoscreen a soybean cDNA library in lambda gt11 and isolate a partial clone of the clathrin heavy chain (HC) gene. Full-length cDNA for soybean clathrin HC was deduced by 5' and 3' cDNA amplification. The cDNA encodes an amino acid sequence of 1,700 residues, which is slightly larger than rat clathrin HC and may account for the reduced mobility of plant clathrin on SDS-PAGE. Insertion of these extra residues is largely confined to the amino and carboxy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are relatively well conserved between eukaryotes. A computer algorithm to determine alpha-helical coiled-coil structures reveals that only one domain, aligning to residues 1,460-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequences. This provides further evidence that the interaction between clathrin heavy and light chains is mediated by three bundles of coiled-coils near to the carboxy terminus. In analysing the role of plant clathrin in endocytotic trafficking, as against trafficking from the Golgi apparatus to the vacuole, our attention was focused on membrane recycling in tip-growing pollen tubes. These rapidly growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled state, suggesting a highly dynamic trafficking pathway. Immunofluorescence analysis of pollen tubes revealed that clathrin localises to the plasma membrane at the apex of the pollen tube tip, which is consistent with high levels of clathrin-mediated membrane recycling. The use of these reagents in conjunction with tip-growing pollen tubes has created a unique opportunity to examine the basis for constitutive endocytosis, so that the more complex question of receptor-mediated pathways in plants can also be assessed.

Amino Acid Sequence↗
PubMed1991

Sequence of critical events involved in fusion of phospholipid vesicles induced by clathrin.

Membrane fusion induced by clathrin is accompanied by several events such as conformational change, membrane binding and association of clathrin, and membrane aggregation (Maezawa et al. (1989) Biochemistry 28, 1422-1428; Maezawa and Yoshimura (1990) Biochem. Biophys. Res. Commun. 173, 134-140). To clarify the sequence of these events, we examined their time-courses by reducing the pH of the medium from 7.4 to a given pH in the range of 3.5-5.0 at 25 degrees C or 10 degrees C. Large unilamellar vesicles composed of phosphatidylserine and phosphatidylcholine were used in most experiments. The half-time for conformational change of clathrin was less than those for membrane binding and association of clathrin. The half-times and the initial rates of membrane binding and association of clathrin were similar order of magnitude, although the pH-profiles of the initial rates of the two events were somewhat different. Membrane aggregation started after membrane binding of clathrin. A lag phase was observed in the time-course of membrane fusion, whereas there was no lag phase in membrane binding and association of clathrin and membrane aggregation. Moreover, the lag time before fusion was independent of the clathrin concentration, although the initial rates of these three events were dependent on it, suggesting that the three reactions are not responsible for the lag phase before fusion, and that there is some other event(s) in the lag time. On the other hand, there was a threshould-pH in the pH profile of the lag-time and the threshold-pH coincided with the critical pH at which the final associated state of clathrin was apparently reversed in the presence and absence of liposomes, suggesting that the event(s) in the lag phase may be related to this final associated state of clathrin molecules on the liposome membranes. These results indicate that clathrin-induced fusion of liposomes is initiated through the following sequential events: conformational change of clathrin, membrane binding and association of clathrin, which occur simultaneously but independently, membrane aggregation, an event(s) in the lag phase, and actual fusion.

Clathrin↗
PubMed2003

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

Amino Acid Motifs↗
PubMed1999

Adaptor complex-independent clathrin function in yeast.

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

Adaptor Proteins, Vesicular Transport↗
PubMed1982

Brain clathrin: studies of its ultrastructural assemblies.

Clathrin purified to a high degree of homogeneity showed the presence of accompanying polypeptides of lower molecular weights and assembled into baskets or cages when the pH of its solution was adjusted from pH 7.5 to 6.5. Brief chymotrypsin treatment of this clathrin preparation at pH 6.5 cleaved clathrin-associated proteins rendering clathrin unable to reform baskets. Hydrolysis of clathrin-associated proteins did not affect clathrin's capacity to polymerize into open lattices or to bind actin and alpha-actinin from solution. When open lattices formed, the turbidity of the solution rose to levels that were higher than those produced when cages or baskets were formed by native clathrin. In both native or enzyme-treated clathrin, turbidity increase was inhibited by salts. The addition of certain cytoskeletal-disrupting agents, such as chlorpromazine and imipramine, increased clathrin's turbidity but did not result in formation of the baskets. Colchicine and cytochalasin B did not affect turbidity or the assembly of cages. The addition of increasing amounts of vinblastine resulted in the precipitation of larger amounts of clathrin which, after solubilization, retained its ability to polymerize into baskets. It seems that clathrin exists as a complex with other protein molecules, clathrin-associated proteins, that modulate the extent of polymerization and assembly the clathrin into characteristic structures.

Actinin↗
PubMed2000

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Animals↗
PubMed2005

Clathrin is required for the function of the mitotic spindle.

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

Animals↗
PubMed2000

A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

Adaptor Protein Complex alpha Subunits↗
PubMed2005

Highly cooperative control of endocytosis by clathrin.

Clathrin assembles into a dynamic two-dimensional lattice on the plasma membrane where it plays a critical role in endocytosis. To probe the regulation of this process, we used siRNA against clathrin, in combination with single cell assays for transferrin uptake as well as total internal reflection microscopy, to examine how endocytic rates and membrane dynamics depend upon cellular clathrin concentration ([Clathrin]). We find that endocytosis is tightly controlled by [Clathrin] over a very narrow dynamic range such that small changes in [Clathrin] can lead to large changes in endocytic rates, indicative of a highly cooperative process (apparent Hill coefficient, n > 6). The number of clathrin assemblies at the cell surface was invariant over a wide range of [Clathrin]; however, both the amount of clathrin in each assembly and the subsequent membrane dynamics were steeply dependent on [Clathrin]. Thus clathrin controls the structural dynamics of membrane internalization via a strongly cooperative process. We used this analysis to show that one important regulator of endocytosis, the actin cytoskeleton, acts noncompetitively as a modulator of clathrin function.

Actins↗
PubMed2005

Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis.

Previously, we reported that the hetero-tetrameric adaptor complex AP-2 co-localizes with the static population of clathrin spots, whereas it is excluded from clathrin spots that disappear from the plasma membrane (forming clathrin-coated vesicles). More recently however, another group provided evidence that AP-2 markers could be observed coincident with disappearing clathrin spots. Thus, we tested several possible explanations for the apparent discrepancies in these two studies. We evaluated the potential contribution of nonred emission of clathrin-dsRed (used in both studies) in the simultaneous measurement of AP-2 and clathrin at various times. Additionally, we directly compared two different green fluorescent protein-tagged AP-2 constructs (similar to those used in the previous reports). These studies demonstrated that the duration of expression time greatly influences the subcellular localization of the AP-2 markers. Furthermore, we quantitatively evaluated the AP-2 fluorescence at the sites of numerous static and disappearing clathrin spots (at least 80 per group) and confirmed our initial observation that while AP-2 is present in nearly all static clathrin spots, it is excluded from the disappearing population of clathrin spots. Finally, in order to verify that clathrin spot disappearance represents clathrin-coated vesicle internalization, we simultaneously imaged clathrin and the cargo molecule transferrin at the cell surface.

Biomarkers↗
PubMed1998

Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.

Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin self-assembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of high-affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation.

Amino Acid Sequence↗
PubMed2000

The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast.

BACKGROUND: In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated. RESULTS: The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain. CONCLUSIONS: These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.

Amino Acid Motifs↗
PubMed1999

An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters.

Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that approximately 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8. Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al. (1997) EMBO J. 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.

Animals↗
PubMed2004

Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy.

Most eukaryotes utilize a single pool of clathrin to assemble clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow clathrin dynamics close to the cell surface, we find that the majority of labeled clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the alpha subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 beta2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile clathrin puncta may be distinct from plasma membrane-associated clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of clathrin structures.

Cell Line↗
PubMed1988

Deep-etch visualization of proteins involved in clathrin assembly.

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.

Aluminum Silicates↗
PubMed1994

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.

Acid Phosphatase↗
PubMed1984

Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

Adenosine Triphosphatases↗
PubMed1999

The majority of clathrin coated vesicles from lactating rabbit mammary gland arises from the secretory pathway.

Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on (2)H2O-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9+/-10.5 nm and a density of 1.23 g x ml(-1). Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only approximately 5-10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents approximately 70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway. Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of alpha(s1)-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.

Adaptor Protein Complex alpha Subunits↗