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S L Schmid

Publications and source records attributed to S L Schmid.

71 records · Page 4Linked to original sources
PubMed1991

Stage-specific assays for coated pit formation and coated vesicle budding in vitro.

Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.

Adenosine Triphosphate↗
PubMed1990

Intracellular transport of class II MHC molecules directed by invariant chain.

Three structural motifs in the invariant chain (li) control the intracellular transport of class II major histocompatibility complex molecules. An endoplasmic reticulum retention signal in the full-length li suggests a role for li in the alpha-beta heterodimer assembly. Another signal motif directs a truncated li, alone or associated with individual class II chains, to a degradation compartment by a pathway circumventing the Golgi. When this truncated li binds alpha-beta dimers, a third signal dominates, directing the complex by way of the Golgi to vesicles in the cell periphery, which may represent a subcompartment of recycling endosomes.

Antibodies, Monoclonal↗
PubMed1990

ATP is required for receptor-mediated endocytosis in intact cells.

We have demonstrated a requirement for cellular ATP in the receptor-mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric-transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I-BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.

Adenosine Triphosphate↗
PubMed1988

Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes.

Functionally distinct subpopulations of endosomes involved in targeting internalized material to specific intracellular destinations were resolved as two distinct peaks by free-flow electrophoresis. The less anodally shifted peak contained a population of early endosomes selectively labeled by brief exposures to endocytic tracers or by receptor-bound transferrin. Late endosomes, labeled only after longer periods of internalization, migrated more toward the anode. While both fluid phase and certain receptor-bound markers could be rapidly chased from early to late endosomes, transferrin remained in vesicles comigrating with early endosomes even after prolonged uptake. Thus early and late endosomes are kinetically related but functionally distinct: early endosomes serve as the major site of recycling of membrane and surface receptors and late endosomes are involved in delivery to lysosomes. The subpopulations each contain unique polypeptides not found on the plasma membrane. Thus, endosomes cannot be derived entirely from internalized cell surface components, and may have at least partly independent biosynthetic origins.

Animals↗
PubMed1988

Isolation of functionally distinct endosome subpopulations by free-flow electrophoresis.

We have described a novel analytical procedure for the one-step purification of endosome subpopulations based on their differing electrophoretic mobilities when subjected to free flow electrophoresis. This procedure offers several advantages over existing schemes for endosome purification. Highly enriched endosomes are recovered in high yield and remain intact, facilitating biochemical and functional analysis of endosome subpopulations. It is rapid, requiring less than 2 hours to complete, and highly reproducible, thus facilitating detailed kinetic analysis of ligand transport through endosomal subpopulations. This technique can be easily scaled up to any degree enabling preparation of large quantities of early and late endosomes for further biochemical analysis and for antibody production. Our results have demonstrated the existence of at least two functionally and biochemically distinct, but kinetically related, subpopulations of endosomes.

Affinity Labels↗
PubMed1987

The ATPase core of a clathrin uncoating protein.

Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.

Adenosine Triphosphatases↗
PubMed1985

Enzymatic dissociation of clathrin cages in a two-stage process.

Uncoating ATPase catalyzes the ATP-dependent dissociation of clathrin from coated vesicles and empty cages. Following an uncoating reaction, clathrin triskelions are released intact, in a stoichiometric complex with bound uncoating protein. This overall uncoating process was dissected into two partial reactions. In the first, ATP hydrolysis drives the transient displacement of a portion of a triskelion from a cage. Uncoating protein then captures the displaced triskelion, in the second stage, by binding to a newly exposed site on clathrin that had previously been buried in the cage lattice. Triskelion-uncoating protein complexes are released when all points of attachment of the triskelion to the cage have been severed. The uncoating protein interacts with a distinct site on clathrin for each of these reactions.

Adenosine Triphosphatases↗
PubMed1985

Two classes of binding sites for uncoating protein in clathrin triskelions.

Clathrin released from coated vesicles or empty cages by the ATP-dependent action of uncoating protein exists as a complex with the uncoating protein. Despite its apparent consumption during a round of uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously recycles from its product (triskelions) to its substrate (cages). The binding of uncoating protein to clathrin triskelions is a complex equilibrium that involves the interaction of uncoating protein with at least two distinct sites on the clathrin molecule. Limited proteolysis dissected clathrin into two domains, each of which contained distinct binding sites. Binding to one of these sites, located on the proximal leg of a triskelion, was dependent upon the presence of light chains and was unstable to gel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, was stable to gel filtration and was independent of the presence of light chains.

Adenosine Triphosphatases↗
PubMed1985

ATP catalyzes the sequestration of clathrin during enzymatic uncoating.

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.

Adenosine Triphosphate↗
PubMed1984

An enzyme that removes clathrin coats: purification of an uncoating ATPase.

Uncoating ATPase, an abundant 70,000-mol-wt polypeptide mediating the ATP-dependent dissociation of clathrin from coated vesicles and empty clathrin cages, has been purified to virtual homogeneity from calf brain cytosol. Uncoating protein is present in cells in amounts roughly stoichiometric with clathrin. This enzyme is isolated as a mixture of monomers and dimers, both forms being active. ATP can support protein-facilitated dissociation of clathrin at micromolar levels; all other ribotriphosphates as well as deoxy-ATP are inactive. The clathrin that is released from cages consists of trimers (triskelions) in a stoichiometric complex with uncoating ATPase. These complexes with clathrin have little tendency to self-associate at neutral pH, and at acidic pH they interfere with the assembly of free clathrin. The possible existence and function of these complexes as clathrin carriers in cells would explain why uncoating protein is made in quantities equivalent to clathrin.

Adenosine Triphosphatases↗
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↗
PubMed1982

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes. Influence of Ca2+ and Mg2+.

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca2+ and Mg2+ on these preferences, have been examined employing 31P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca2+ and Mg2+. In mixed systems with (HII phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (less than 10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca2+ (but not Mg2+) can induce HII phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca2+ induces structural reorganizations in mixed systems containing acidic phospholipids and phosphatidylethanolamine.

Calcium↗
PubMed1982

A domain of clathrin that forms coats.

Triskelions, trimeric complexes of clathrin and associated light chains, are the assembly units of clathrin coats [Ungewickell, E. & Branton, D. (1981) Nature (London) 289, 420-422; Kirchhausen, T. & Harrison, S. C. (1981) Cell 23, 755-761]. We report here that triskelions whose outer arms have been removed by trypsin digestion retain the ability to be assembled into coats. These digested timers contain a 110,000 molecular weight domain of clathrin and lack intact light chains.

Animals↗
PubMed1980

Phospholipids and membrane transport.

The ability of membrane lipids to adopt nonbilayer configurations suggests dynamic roles for lipids in many functional abilities of biological membranes. In this work evidence supporting the involvement of lipids in three types of membrane transport process is presented and discussed. These transport processes include facilitated transbilayer transport of polar molecules, transport mechanisms involving fusion events, and transport possibilities arising from alternative membrane morphology. In particular it is shown that lipids such as cardiolipin, which adopt the hexagonal HII phase in the presence of Ca2+, may be logically proposed to facilitate Ca2+ transport across membranes via an inverted micellar intermediate. Alternatively, in transport processes such as exocytosis the ability of Ca2+ to generate membrane instabilities favouring nonbilayer alternatives suggests a crucial role of phospholipid in the fusion event vital to exocytotic release. Finally, nonbilayer lipid structures may be suggested to favour formation of isolated compartments connected by a continuous membrane where lateral diffusion processes can lead to transport. These various possibilities are summarized in a "metamorphic mosaic" model of biological membranes.

Adrenal Glands↗
PubMedDate not supplied

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation.

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Adaptor Protein Complex 2↗
PubMedDate not supplied

A role for clathrin light chains in the recognition of clathrin cages by 'uncoating ATPase'.

A cycle of clathrin assembly and disassembly drives the formation of coated vesicles, intermediates in intracellular protein transport. The heavy chain of clathrin is needed for assembly, but the function of the clathrin light chains has remained obscure. An enzyme has now been purified which uses ATP hydrolysis to power the release of clathrin from coated vesicles, presumably recycling the coat protein for repeated rounds of vesicle budding. This 'uncoating ATPase' requires clathrin light chains for its action.

Adenosine Triphosphatases↗