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

301

190

Abstract. Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is *10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be •20 % as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

Section: Discussionmentioning

confidence: 99%

Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

Jing

Spencer

Miller

et al. 1990

301

190

“…Clathrin and adaptors were purified using a method based on that of Manfredi and Bazari (1987) with the modifications described by Chang et al (1993). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated in the text.…”

Section: Methodsmentioning

confidence: 99%

In vivo phosphorylation of adaptors regulates their interaction with clathrin.

Wilde

Brodsky

1996

146

148

Abstract. 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, et and 132 of AP2 and 131 of AP1, but not the ~/subunit of AP1. In addition, the ix1 and p.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 t32 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. C LATHRIN-MEDIATED vesicular transport occurs at two sites within the cell. At the plasma membrane, clathrin-coated vesicles (CCV) 1 facilitate receptor-mediated endocytosis (Pearse and Robinson, 1990) to internalize soluble extracellular molecules such as nutrients, antigens, and growth factors and to down-regulate their receptors. CCV are also formed at the TGN where they sort newly synthesized lysosomal enzymes to the endocytic pathway and play a role in secretory granule biogenesis (Pearse and Robinson, 1990). The CCV coat is formed by polymerization of triskelion-shaped clathrin molecules into a lattice, catalyzed by adaptor molecules. The AP2 and AP1 adaptors nucleate clathrin assembly at the plasma membrane and the TGN, respectively, and play a role in selecting integral membrane proteins for

“…Immunoprecipitations were carried out on HeLa cell extracts under nondenaturing conditions, as previously described (Hirst et al, 1999;Page et al, 1999). Clathrin-coated vesicles were purified from rat liver as described by Manfredi and Bazari (1987). For gel filtration, pig brain cytosol was prepared by homogenizing 1 g pieces of pig brain in 2 ml cytosol buffer (25 mM Hepes-KOH, pH 7.0, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM dithiothreitol and 1 mg/ml glucose) and fractionated on a Superose 6 100-ml column as described by Stamnes and Rothman (1993).…”

Section: Immunoprecipitations and Western Blottingmentioning

confidence: 99%

A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome

Hirst

Lui

Bright

et al. 2000

317

388

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal “ear” domain of γ-adaptin. However, unlike γ-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the γ-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.