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F M Brodsky

Publications and source records attributed to F M Brodsky.

At least 19 recordsLinked to original sources
PubMed1999

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

Amino Acid Sequence↗
PubMed1999

Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression.

Viral strategies for immune evasion include inhibition of various steps in the class I MHC assembly pathway. Here, we demonstrate that adenovirus produces one gene product with a dual function in this regard. It is well established that adenovirus E19 binds class I molecules and retains them in the endoplasmic reticulum (ER). However, E19 also delays the expression of class I alleles to which it cannot tightly bind. Here, we show that E19 binds TAP and acts as a tapasin inhibitor, preventing class I/TAP association. DeltaE19, an E19 mutant lacking the ER-retention signal, delays maturation of class I molecules, indicating that E19's inhibition of class I/TAP interaction is sufficient to delay class I expression. These data identify tapasin inhibition as a novel mechanism of viral immune evasion and suggest that, through this secondary mechanism, adenovirus can affect Ag presentation by MHC alleles that it can only weakly affect by direct retention.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake.

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Amino Acid Sequence↗
PubMed1999

Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases.

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.

Antigens, CD↗
PubMed1999

Human pathogen subversion of antigen presentation.

Many pathogens have co-evolved with their human hosts to develop strategies for immune evasion that involve disruption of the intracellular pathways by which antigens are bound by class I and class II molecules of the major histocompatibility complex (MHC) for presentation to T cells. Here the molecular events in these pathways are reviewed and pathogen interference is documented for viruses, extracellular and intracellular bacteria and intracellular parasites. In addition to a general review, data from our studies of adenovirus, Chlamydia trachomatis and Coxiella burnetii are summarized. Adenovirus E19 is the first viral gene product described that affects class I MHC molecule expression by two separate mechanisms, intracellular retention of the class I heavy chain by direct binding and by binding to the TAP transporter involved in class I peptide loading. Coxiella and Chlamydia both affect peptide presentation by class II MHC molecules as a result of their residence in endocytic compartments, although the properties of the parasitophorous vacuoles they form are quite different. These examples of active interference with antigen presentation by viral gene products and passive interference by rickettsiae and bacteria are typical of the strategies used by these different classes of pathogens, which need to evade different types of immune responses. Pathogen-host co-evolution is evident in these subversion tactics for which the pathogen crime seems tailored to fit the immune system punishment.

Animals↗
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↗
PubMed1998

A novel AP180-related protein in vesicles that concentrate at acetylcholine receptor clusters.

Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305-598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with gamma adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway.

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late endocytic compartments.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Interactions between HIV1 Nef and vacuolar ATPase facilitate the internalization of CD4.

CD4 is the primary receptor for the human immunodeficiency virus (HIV). Nef is an accessory protein of HIV that decreases the expression of CD4 on the surface of infected cells. In this study, we identified the Nef binding protein 1 (NBP1), which interacts specifically with Nef in vitro and in vivo. Since it shares sequence similarity with the catalytic subunit of the vacuolar ATPase (V-ATPase) and complements the loss of this VMA13 gene in yeast, NBP1 is the human homolog of Vma13p. Direct interactions between Nef and NBP1 were correlated with the ability of Nef to internalize CD4. The expression of the antisense NBP1 abrogated these effects. We conclude that NBP1 helps to connect Nef with the endocytic pathway.

Amino Acid Sequence↗
PubMed1997

Localization of class I histocompatibility molecule assembly by subfractionation of the early secretory pathway.

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with beta 2-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 degrees C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected suggesting empty molecules recycle to acquire peptide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

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.

Adaptor Protein Complex 1↗
PubMed1996

A membrane-associated protein complex with selective binding to the clathrin coat adaptor AP1.

Adaptors are the membrane-binding components of clathrin-coated vesicles. The interaction of the trans-Golgi coat adaptor AP1 with membrane-associated proteins was analyzed by affinity chromatography. Proteins of 83 and 52 kDa bound specifically to the core domain of AP1 and showed no interaction with AP2 or other clathrin-coated vesicle proteins. The AP1-binding proteins were tightly membrane-associated, though behaved as peripheral membrane proteins. They were detected in membranes depleted of clathrin-coated vesicles and not in coated vesicles, suggesting that the interaction of these proteins with AP1 may precede coated vesicle budding. Co-fractionation of the AP1-binding proteins with trans-Golgi network membrane was also observed. Upon gel filtration, both AP1-binding proteins eluted in a high molecular mass complex which was labile at high concentrations of Tris. The 83 kDa protein bound to AP1 affinity resin in the absence of the 52 kDa protein. In contrast, the separated 52 kDa protein did not bind AP1, suggesting that the 83 kDa protein is the AP1-binding component of the complex. Characterization of this protein complex defines a novel membrane-associated component that specifically interacts with AP1 and may contribute to its function in forming clathrin-coated vesicles.

Adaptor Protein Complex 1↗
PubMed1995

Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs.

Clathrin polymerization into a polyhedral vesicle coat drives receptor sorting at cellular membranes during endocytosis and organelle biogenesis. To study clathrin self-assembly, we expressed the C-terminal third of the clathrin heavy chain in bacteria. The recombinant fragment trimerized, bound clathrin light chains, and morphologically resembled the hub domain of the triskelion-shaped clathrin molecule. Self-assembly of recombinant hubs demonstrated a regulatory role for clathrin light chains and for the distal portions of triskelion legs in clathrin coat formation. Deletion mutagenesis of the hub localized a domain mediating light chain binding and clathrin self-assembly and mapped a transferable trimerization domain. These studies define molecular interactions controlling clathrin self-assembly and establish a recombinant system for future analysis.

Animals↗
PubMed1995

The interaction of calmodulin with clathrin-coated vesicles, triskelions, and light chains. Localization of a binding site.

The binding of clathrin-coated vesicles, clathrin triskelions, and free clathrin light chains to calmodulin-Sepharose was compared. When isolated from bovine brain, all three components bound to calmodulin-Sepharose in the presence of calcium and could be eluted by its removal. In contrast, coated vesicles and triskelions isolated from bovine adrenal gland did not bind to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain. As distinct isoforms of the clathrin light chains are expressed by brain and adrenal gland, these results implicate the clathrin light chains as the calmodulin-binding component of coated vesicles and triskelions. Furthermore, the insertion sequences found in the neuron-specific isoforms, although not necessary for the binding of free clathrin light chains to calmodulin, must facilitate the interaction of heavy chain-associated light chains with calmodulin. Recombinant mutants of LCa, with deletions spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity, as assessed by the binding of a panel of monoclonal antibodies, exhibited varying amounts of calmodulin binding activity. However, deletion of the carboxyl-terminal 20 residues abolished calmodulin interaction. Thus, the carboxyl terminus of LCa appears to constitute a calmodulin-binding site. Peptides corresponding to the carboxyl terminus of LCa or LCb inhibited the interaction of the light chains with calmodulin, suggesting that this region forms the calmodulin-binding site of both LCa and LCb. The carboxyl-terminal peptides of LCa and LCb inhibited the interaction of light chains with calmodulin approximately 10-fold less effectively than a calmodulin-binding peptide derived from smooth muscle myosin light chain kinase, but much more effectively than a calmodulin-binding peptide derived from adenylate cyclase. This comparison places the clathrin light chain-calmodulin interaction within the physiological range seen for other calmodulin-binding proteins.

Adrenal Glands↗
PubMed1993

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

Adaptor Protein Complex 2↗