Isolation and Preliminary Characterization of Clathrin-Associated Proteins

Lisanti, Michael P.; Shapiro, Lawrence; Moskowitz, Nathan; Hua, Elaine L.; Puszkin, Saul; Schook, William

doi:10.1111/j.1432-1033.1982.tb06706.x

Cited by 78 publications

(38 citation statements)

References 19 publications

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“…2). Clathrincoated vesicles are known to be associated with a large variety of proteins in addition to transferrin (28). Thus some mobilferrin was present in fractions containing the clathrin immunoreactive fractions which are associated with the transferrin containing vesicle.…”

Section: Resultsmentioning

confidence: 88%

Mobilferrin is an intermediate in iron transport between transferrin and hemoglobin in K562 cells.

Conrad

Umbreit²,

Moore³

et al. 1996

J. Clin. Invest.

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Iron is bound to transferrin in the plasma. A specific receptor on the cell surface binds transferrin and internalizes transferrin and the iron in clathrin-coated pits. These invaginate to form vesicles which release iron to the cytoplasm. Inorganic iron can be transported by an alternative pathway from iron citrate, utilizing a cell surface integrin and a cytoplasmic protein mobilferrin. This article shows that the two pathways donate iron to mobilferrin which acts as an intermediate between the iron bound to transferrin and the incorporation of iron into hemoglobin. Mobilferrin is found associated with the transferrin containing vesicles, and becomes labeled with iron released from transferrin in the vesicles. Mobilferrin is also found in the cytoplasm where pulse-chase experiments show that it, in turn, releases iron to be used for the synthesis of hemoglobin. ( J. Clin. Invest.

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Section: Resultsmentioning

confidence: 88%

Mobilferrin is an intermediate in iron transport between transferrin and hemoglobin in K562 cells.

Conrad

Umbreit²,

Moore³

et al. 1996

J. Clin. Invest.

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“…A concentrated light chain sample was prepared as described by Lisanti et al (1982b) by incubating 1.5 ml of clathrin (9 mg/ml) at 95°C for 10 min. After removal of the heavy chain precipitate by a 5 min spin in an Eppendorf centrifuge, -1.2 mg of light chains were recovered in the supernatant.…”

Section: Separation Of Heavy and Light Chainsmentioning

confidence: 99%

Clathrin heavy chain, light chain interactions.

Winkler¹,

Stanley²

1983

The EMBO Journal

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Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non‐denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one‐dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(‐10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures.

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“…Clc exhibits several properties that suggest it may act as a regulatory subunit (4). In vitro, Clc can bind calmodulin, Hsc70, and calcium (5)(6)(7). LC b can be phosphorylated in vitro by casein kinase II and is phosphorylated at the same sites in vivo (8).…”

mentioning

confidence: 99%

The Light Chain Subunit Is Required for Clathrin Function in Saccharomyces cerevisiae

Chu¹,

Pishvaee

Payne

1996

Journal of Biological Chemistry

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Clathrin, a multimeric protein involved in intracellular protein trafficking, is composed of three heavy chains (Chc) and three light chains (Clc). Upon disruption (clc1⌬) of the single Clc-encoding gene (CLC1) in yeast, the steady state protein levels of Chc decreased 5-10-fold compared with wild type cells; consequently, phenotypes exhibited by clc1⌬ cells may result indirectly from the loss of Chc as opposed to the absence of Clc. As an approach to directly examine Clc function, clc1⌬ strains were generated that carry a multicopy plasmid containing the clathrin heavy chain gene (CHC1), resulting in levels of Chc 5-10-fold elevated over wild-type levels. As with deletion of CHC1, deletion of CLC1 results in defects in growth, receptor-mediated endocytosis, and maturation of the mating pheromone ␣-factor. However, elevated Chc expression in clc1⌬ cells partially suppresses the growth and ␣-factor maturation defects displayed by clc1⌬ cells alone. Biochemical analyses indicate that trimerization and assembly of Chc are perturbed in the absence of Clc, resulting in vesiculation defects. Our results demonstrate that the light chain subunit of clathrin is required for efficient Chc trimerization, proper formation of clathrin coats, and the generation of clathrin-coated vesicles.Distinct compartments in eukaryotic cells are maintained through the selective transport of proteins carried out by small vesicular carriers. Generation of these vesicles involves assembly of proteinaceous coats on the cytoplasmic surface of donor compartment membranes, which leads to the budding of coated vesicles. Although a variety of proteins are transported through vesicular movement, a subset of specific trafficking events are mediated by clathrin-coated vesicles. These include the retention of resident Golgi membrane proteins, receptormediated endocytosis, and the sorting of lysosomal/vacuolar proteins from the secretory pathway to the lysosome/vacuole (1, 2).The clathrin molecule, or triskelion, is a hexamer composed of three heavy chain subunits (Chc) and three light chain subunits (Clc) (1). In mammals, there are two forms of light chain, LC a and LC b , which share 60% amino acid identity and appear to be randomly distributed in clathrin trimers. In yeast, there is only one form of light chain encoded by the CLC1 gene (3). Formation of a clathrin-coated vesicle is initiated by binding of clathrin-associated protein complexes (APs) 1 to the donor membrane. Triskelions associate with the APs and polymerize into polygonal lattice structures. Such clathrin-coated membrane segments, known as coated pits, are thought to collect specific cargo proteins through interactions between the cargo protein cytoplasmic domains and the AP complexes. The clathrin-coated pit then invaginates and pinches off to form a clathrin-coated vesicle carrying the selected cargo proteins. Formation of the vesicle may involve rearrangement of subunits assembled on the coated pit, or it may be driven by the polymerization of new clathrin subunits into a polyhe...

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Isolation and Preliminary Characterization of Clathrin-Associated Proteins

Cited by 78 publications

References 19 publications

Mobilferrin is an intermediate in iron transport between transferrin and hemoglobin in K562 cells.

Mobilferrin is an intermediate in iron transport between transferrin and hemoglobin in K562 cells.

Clathrin heavy chain, light chain interactions.

The Light Chain Subunit Is Required for Clathrin Function in Saccharomyces cerevisiae

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