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PubMed2006

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Adaptor Protein Complex 2↗
PubMed1999

Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.

The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.

Amino Acids↗
PubMed1994

Amyloid precursor protein is enriched in axolemma and periaxolemmal-myelin and associated clathrin-coated vesicles.

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal-myelin from rat brain and have determined by Western blot that APPs, Mr 100-110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal-myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal-myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti-C-terminal APP antibodies but not with anti-N-terminal antibody, appears to be a membrane-associated C-terminal fragment. Western blots with antibodies to Alzheimer precursor-like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100-110 and the 10.5 kDa C-terminal peptide. The demonstration of APP in axolemma and periaxolemmal-myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin-coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal-myelin. In addition, these vesicles contain cysteinyl and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100-110 and Mr 10.5 when analyzed with antibodies to APP672-695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C-terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C-terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C-terminus and may be a useful tool for altering C-terminal APP function.

Amino Acid Sequence↗
PubMed1996

Desensitization and internalization of adenosine A1 receptors in rat brain by in vivo treatment with R-PIA: involvement of coated vesicles.

Chronic treatment of rats with R-PIA 'in vivo' desensitized adenosine A1 receptor-mediated inhibition of adenylyl cyclase in brain plasma membranes and increased basal and forskolin-stimulated adenylyl cyclase. The adenosine A1 receptor agonist CHA (cyclohexyl adenosine) inhibited forskolin-stimulated adenylyl cyclase in synaptic plasma membranes from control rats but failed to do so in membranes isolated from rats treated with R-PIA. This loss of response was accompanied with a significant decrease in both, total number of adenosine A1 receptors and steady-state level of alpha-Gi in synaptic plasma membranes. An increase in the steady-state level of alpha-Gs in synaptic plasma membranes was also observed by R-PIA treatment. Concurrently, a significant increase of adenosine A1 receptors was observed in microsomes and coated vesicles. These results demonstrate adenosine A1 receptor desensitization in rat brain by 'in vivo' treatment with R-PIA and suggest a role for coated vesicles in the internalization of G-protein coupled receptors.

Adenosine↗
PubMed1995

Distinct coated vesicles labeled for p200 bud from trans-Golgi network membranes.

Golgi-associated cytoplasmic proteins, such as the coatomer protein complex, are required for vesicle budding and trafficking. We have previously described a cytoplasmic phosphoprotein, p200, which binds dynamically and specifically to Golgi membranes. The p200 protein is dissociated from Golgi membranes in the presence of brefeldin A and it is induced to bind to Golgi membranes by activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-[gamma-thio]triphosphate or aluminum fluoride. To establish the role of p200 in vesicle budding, we localized membrane-bound p200 in intact cells and on isolated Golgi membranes. We show that p200 is preferentially associated with vesicles in the trans-Golgi network (TGN). Activation of G proteins induced budding and accumulation of small, coated vesicles from Golgi membranes and p200 was localized on the cytoplasmic surface of some of these vesicles. Using immunogold labeling we further demonstrate that p200 and beta-COP are localized on different populations of Golgi-derived vesicles. These data establish that p200 is involved in the budding and coating of a class of Goli vesicles that are likely to be derived from the TGN. The data also show that there are distinct populations of non-clathrin-coated vesicles budded from Golgi membranes, and vesicles labeled for either beta-COP or p200 may represent transport vesicles for separate steps of protein transport.

Aluminum Compounds↗
PubMed1982

Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.

When 125I-labeled insulin (125I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, 125I-insulin localizes to larger intracellular membrane-bounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of 125I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.

Animals↗
PubMed2003

Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers.

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50-60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.

Animals↗
PubMed1991

Detection of G proteins in bovine brain clathrin coated vesicles with common alpha and beta subunits antibodies.

Clathrin coated vesicles (CV) from bovine brain were analyzed via Western blots for the presence of the alpha and beta subunits of guanine nucleotide regulatory proteins. The results with the common alpha antibody GA/1 revealed the presence of apparently undissociated G-protein subunits migrating at approximately 80 kDa. The predominant band was of about 39-41 kDa, with minor labeling observed in the 41-52 kDa range. Western blot analysis for G beta subunit(s) revealed the presence of a single band of about 35-36 kDa.

Animals↗
PubMed1988

Molecular cloning and complete amino acid sequence of AP50, an assembly protein associated with clathrin-coated vesicles.

AP50 is the 50,000-dalton protein component found in clathrin-coated vesicles as part of the coat assembly protein (AP) complex, AP-2. AP50 cDNA clones were isolated from rat brain cDNA libraries, and their nucleotide sequence was determined. The isolated cDNA clones represent the entire coding sequence for the rat brain AP50. They encode a polypeptide containing 435 amino acids with a molecular weight of 49,612 daltons. Comparison with the partially sequenced bovine brain AP50 shows a primary structure that is highly conserved. AP50 does not have detectable sequence similarity with other known kinases or with other proteins of known sequence.

Adaptor Protein Complex 2↗
PubMed1992

Coated vesicles are implicated in the post-fusion retrieval of the membrane of rat atrial secretory granules.

Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release.

Animals↗
PubMed1996

Sorting by COP I-coated vesicles under interphase and mitotic conditions.

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

Amino Acid Sequence↗
PubMed1976

A three-dimensional reconstruction of the polygonal pattern on placental coated-vesicle membranes.

There is a surface structure on the coated vesicles of human placenta. Some features of this structure have been examined. Measurements of the polygonal network seen in surface views have been made and compared with measurements of structures projecting from vesicle walls in median sections. A 3-dimensional reconstruction of the vesicle shows the pattern to be one of raised ridges. Use of a goniometer to tilt the specimens has confirmed the assumption that both types of image obtained as from one structure. Although it is usually the case that vesicles are approximately spherical, some are definitely irregularly shaped. For this reason it is suggested that the walls of the polygons need not always be packed into a structure with a regular and precisely maintained pattern. Consideration of the surface structure in the light of current understanding of the cell membrane as a dynamic system leads to a possible explanation of the process of vesicle formation in this context and of the selective nature of uptake by micropinocytosis.

Humans↗
PubMed1983

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles.

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This "burst phase" lasts about 3-5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol. 40, 245-255; D. Epel, 1978, Curr. Top. Dev. Biol. 12, 185-246) that initiation of endocytosis is most likely a Ca2+-dependent event.

Animals↗
PubMed1982

Identification and distribution of coated vesicles in the retinal pigment epithelium of man and rabbit.

Coated pits (CPs) and coated vesicles (CVs) were investigated in the retinal pigment epithelium (RPE) of man and rabbit. In both species CPs and CVs show the typical coat of bristles radiating from their convex surface. CPs originate only from invaginations of the basal and apical cell membrane. The bipolar distribution of CPs is reflected in the distribution of CVs, which are most concentrated near the basal and apical cell boundaries. Neither fusion of CVs with the cytoplasmic organelles nor the presence of receptosomes could be demonstrated. The distribution patterns of CPs, CVs, Golgi apparatus, and mitochondria are very similar in RPE of man and rabbit. Moreover, as compared with other ocular tissues. CVs found in RPE differ both in size and distribution from those found in the pigment epithelium of the pars plana ciliaris and in trabecular endothelial cells. This peculiarity might indicate that receptor-mediated endocytosis via CVs plays a role in the transport across RPE of substances that are specific to this epithelium. Some analogies between the specificity of size and distribution of CVs in RPE and the process of internalization and transport of retinal are suggested.

Animals↗
PubMed2001

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles.

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

Adaptor Protein Complex beta Subunits↗
PubMed1994

En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles.

The cDNA encoding epsilon-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant His6- tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His6 tag. Radiolabeled coatomer was employed to establish that all the subunits of the coatomer enter coated vesicles as an intact unit.

Amino Acid Sequence↗
PubMed1993

Spectroscopical characteristics of galactosylceramide-pyrene and ceramide-pyrene incorporated in model and in clathrin coated vesicles.

In order to control the ability of two pyrene-sphingolipids (ceramide-pyrene (Cpyr) and galactosylceramide-pyrene (GCpyr)) to monitor the changes in the lipid bilayer dynamics of cellular membranes, their incorporation in three populations of clathrin coated vesicles which differ in their structural characteristics (Bomsel et al. (1988) Biochemistry 27, 6808-6812) was studied by both absorbance and fluorescence spectroscopy. The method of injection of an ethanolic solution of probe was used. The analysis of the spectra recorded after injection into a free-membrane buffer allowed to discriminate two dispersion states (micellar or aggregated) of the probes. The micellar state was identified as the one suitable for the incorporation within the bilayer. Rising the temperature up to 18 degrees C for a membrane labeling with GCpyr and to 37 degrees C for a membrane labeling with Cpyr was found to be necessary because it allowed to slow down the aggregation process which inhibited the incorporation within the lipid bilayer. The excimer/monomer (E/M) fluorescence intensities ratio of GCpyr was found to be characteristic of each population of coated vesicles. Cpyr could not be used as a diffusion probe because it partly aggregated during the cooling step necessary to establish the E/M versus temperature plot in the heating mode. An important point which arises from these data is that the use of absorbance spectroscopy can avoid misinterpretation of the pyrene derivatives fluorescence spectra in terms of diffusion.

Buffers↗
PubMed1993

The coated vesicle vacuolar (H+)-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2.

We have previously noted a 50-kDa polypeptide (p50) co-purifying with preparations of the bovine brain clathrin-coated vesicle vacuolar (H+)-ATPase (V-ATPase) (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). We show that p50 is also immunoprecipitated with the V-ATPase, further suggesting its specific association with the proton pump. To determine the identity of this 50-kDa polypeptide and the stoichiometry of its association with the V-ATPase, we performed N-terminal amino acid sequencing and quantitative amino acid analysis of the gel-purified protein. These results revealed the unknown polypeptide to be the 50-kDa subunit of the clathrin assembly protein AP-2 (AP50); we estimate the stoichiometry of association is one AP50 per V-ATPase complex. AP50 is an N-ethylmaleimide (NEM)-inhibitable autokinase and incubation of purified V-ATPase with [gamma-32P]ATP resulted in the NEM-sensitive phosphorylation of AP50 and the B subunit of the V-ATPase. The same phosphorylation pattern is seen if the labeling reaction is done with intact clathrin-coated vesicles and the V-ATPase subsequently immunoprecipitated from the solubilized vesicles. This represents the first report of phosphorylation of one of the V-ATPase subunits. The functional significance of this phosphorylation for regulation or targeting of the V-ATPase in vivo remains to be determined.

Adaptor Protein Complex 2↗