Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane

Clathrin in coated vesicles is linked to transmembrane receptors by adaptor protein complexes. The Golgi-associated adaptor complex HA1 is a tetramer, made up of beta', gamma, 47-kDa, and 20-kDa subunits, whereas the tetrameric plasma membrane adaptor, HA2, contains alpha, beta, 50-kDa, and 16-kDa subunits (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929). Here we report on the structural organization of adaptor subunits as revealed by proteolytic dissection. We show that the beta' and gamma subunits of HA1 are cleaved into 60-67-kDa "trunk" and 32-44-kDa "head" fragments. Interactions between adaptor subunits involve the trunk domains only. In overall organization of their domains, the Golgi and plasma membrane adaptors are very similar. The similarity encompasses also the location of phosphorylated serine residues in the alpha a, beta, beta', and gamma subunits, which are found in the head domains in all cases. In the alpha a and beta subunits they probably occur in the proline- and glycine-rich hinge region, which connects the head to the trunk. Identical adaptor fragments were obtained by controlled digestion of clathrin-coated vesicles. Under conditions that did not affect the integrity of the clathrin heavy chain, the adaptor head fragments were always quantitatively released from coated vesicles. The release of the bulk of the adaptors occurred concomitantly with the cleavage of their beta-type subunits (beta and beta') and under buffer conditions that prevent aggregation of adaptors. These observations taken together with the results of reconstitution experiments confirm and extend previous data (Ahle, S., and Ungewickell, E. (1989) J. Biol. Chem. 264, 20089-20093) which suggested that adaptors attach to clathrin through their beta-type (beta and beta') subunits. Moreover, high affinity interaction between adaptors and clathrin requires the participation of regions from both the head and trunk domains of the beta-type subunits.     

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.     

Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, (1424)YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the mu subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of mu 2. Furthermore, isolated mu 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that mu 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative mu 2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the mu 2 subunit of AP-2 and mutations in mu 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.     

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin‐independent endocytosis, and recently, flotillin‐1 was implicated in the protein kinase C (PKC)‐triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin‐mediated, we re‐examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin‐1, flotillin‐2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC‐dependent internalization and degradation of HA‐DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP‐2 as well as a dynamin inhibitor Dyngo‐4A significantly decreased PKC‐dependent endocytosis of HA‐DAT. Similarly, endocytosis and degradation of DAT that is not epitope‐tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co‐localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA‐DAT in the plasma membrane, suggesting that flotillin‐organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin‐mediated endocytosis is the major pathway of PKC‐dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis .     

Identification of the hyaluronan receptor for endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).     

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.     

Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain

We have identified a new coat protein in clathrin-coated vesicles from bovine brain by urea-SDS gel electrophoresis. The protein was purified from Tris-solubilized coat proteins either by combination of hydroxyapatite chromatography and gel filtration or more rapidly in a single step by immunoaffinity chromatography. The purified protein binds to clathrin triskelia and thereby promotes clathrin assembly into regular 50-100-nm cages. We propose for the new protein the name auxilin (Latin auxilium, meaning support). Auxilin migrates as a 110-kD polypeptide in standard type SDS-PAGE, but in the presence of 6 M urea shifts to a position corresponding to 126 kD. Gel filtration in 6 M guanidinium hydrochloride gives a molecular weight of approximately 86,000. The native protein is monomeric in 0.5 M Tris. Antigenic reactivity and two-dimensional peptide maps gave no evidence of gross similarities between auxilin and any of the other known coated vesicle-associated proteins. Since the structural organization of auxilin does not resemble that of the ubiquitous heterotetrameric HA1 and HA2 adaptor complexes, that are believed to connect clathrin to receptors, it is unlikely that it functions as an adaptor. Immunoblotting did not reveal the presence of auxilin in tissues other than brain. If auxilin and AP 180 are indeed both confined to neuronal cells, as the immunochemical evidence suggests, it might be inferred that both serve to adapt clathrin-coated vesicles to an as yet undisclosed function unique to this cell type.     

Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.     

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.     

Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites

Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.     

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.     

In vivo phosphorylation of adaptors regulates their interaction with clathrin

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self- assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly.     

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.     

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.     

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.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins

Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of alpha subunits of AP-2 adaptor complex to cytosol and this effect was higher in the alpha2 subunit. A high serine phosphorylation status of alpha subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.     

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein.

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.