Cell-cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence of YRSL conforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits. In vitro assays demonstrate that this interaction occurs via the YRSL sequence of L1 and the mu 2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.
Binding sites for antibodies specific for proteins S4 and S14 of the small subunit of E. coli ribosomes have been mapped on the surface of the subunit by electron microscopy. Antibody binding to reconstituted subunits was shown to depend specifically on the presence of E. coli S4 and S14. Anti-S14 IgG was found to bind to a limited region of the ribosome surface. In contrast anti-S4 IgG was found to bind to three separated regions of the ribosome surface, suggesting-S4 has an elongated conformation in situ.In order to understand the molecular basis of the functions performed by the small subunit of the Escherichia coli (E.) ribosome it is necessary to know the arrangement and function of the proteins and RNA in the ribosome. The availability of antibodies specific for individual proteins (1), the development of techniques for electron microscopic visualization of antibodies bound to subunits (refs. 2, 3, 4, and 5), and the interpretation of the two-dimensional images of small subunits seen in electron micrographs as views of a single three-dimensional structure, make possible the location of exposed ribosomal proteins in the three-dimensional structure of the ribosome.In this paper we examine the distributions of binding sites for antibodies specific for two individual ribosomal proteins, S4 and S14, on the surface of the small subunit of E. ribosomes (preliminary report, ref. 5). Anti-S14 IgG (AS14) binding occurred only at one region of the small subunit, while anti-S4 IgG (AS4) binding occurred at three distinct regions of the subunit, indicating that S4 has an elongated structure in situ. MATERIALS AND METHODSE. (strain Q13) ribosomes were prepared according to Iwasaki et al. (6). Subunits were heated for 5 min at 400 in buffer I (10 mM MgCI2, 10 mM Tris HCl, pH 7.8, 200 mM NH4Cl), or for 2 min in buffer II (buffer I except 1 mM MgCl2), then reacted with IgG antibodies for 2 min at 40°and then 20 min at 00. Dimers and monomers of the small subunit were separated from each other and the unreacted antibodies by sedimentation on 15-30% sucrose gradients (3 hr at 234,000 X g, 40, buffer I or buffer II). Sucrose was removed by gel filtration on Sephadex G-100. Alternately, 30S dimers and monomers were. separated from unreacted antibodies byAbbreviations: E., B., obtained from Escherichia coli and Bacillus stearothermophilus, respectively; AS14 and AS4, antibodies to proteins S14 and S4, respectively. 4688 fractionation on a Sepharose 6B column (buffer I or buffer II). Both monomer and dimer fractions were examined by electron microscopy and the dimer fraction was occasionally augmented with part of the monomer peak to obtain optimum concentrations for negative staining.Purification of E. coli and Bacillus 8tearothermophilus (B.) strain 799 ribosomal proteins, reconstitution of 30S subunits, and the assay of their activity were done as previously described (7, 8). The IgG fractions of specific rabbit antisera against purified ribosomal proteins (9) RESULTSSmall subunits were reacted with IgG antibodies a...
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