Ernst Ungewickell

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site.

Binding of AP180 to clathrin triskelia induces their assembly into 60‐70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N‐terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C‐terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1‐20). This protein, localized to synaptic termini, has so far been of unknown function.

A topographical study of the 5'-region of 16 S rna of Escherichia coli in the presence and absence of protein S4.

It was demonstrated, earlier, that the digestion of either 16 S ribosomal RNA, or a complex of protein S4 with the 16 S RNA of Escherichia coli, at very low levels of soluble pancreatic ribonuclease, yields a resistant RNA fragment that sediments at about 9 S [ 1,2] . Oligonucleotide fingerprint analyses of the RNA fragment revealed that it derives from the S’end of the 16 S RNA. This RNA region (S4-RNA) interacts exclusively with protein S4 [ 1,2] . More recently, a similar RNA region was isolated by degrading a protein S4-16 S RNA complex with Tr ribonuclease and the identities of (a) the subfragments contained within the RNA region and (b) the enzyme cutting positions, were established [3] . These RNA subfragments lie within a discontinuous region of sequence, extending from section L to C”, and constitute a total of about 500 nucleotides. Since protein S4 (mol. wt 22 500) is small compared with the S4-RNA site (maximum molecular weight about 165 000), the basis of the protein protection, or stabilization, of the RNA site was assumed to be partly due to the protein, rendering a few critical sequence regions inaccessible to the ribonuclease, and

FURTHER CHARACTERISATION OF THE RNA STRUCTURE IN THE BINDING REGION OF PROTEIN S4 ON 16 S RIBOSOMAL RNA OF ESCHERICHIA COLI

Protein S4 of the Escherichia coli ribosome assembles with a large region of RNA at the .5’-end of the 16 S RNA (reviewed in [l] ). This RNA region (S4-RNA) is exceptional in that it can be organised into a compact structure, thereby creating a binding site for protein S4 (see [2] ). Protein ,‘1, itself, contains two distinct domains, namely the C-terminal three-quarters of the protein and the N-terminal quarter [3-51 . The former domain contains the primary RNA binding region of the protein, and three peptides have been identified in this region that crosslink towards the 3’-end of the RNA region [6-71. The N-terminal domain is probably primarily involved in interacting with other proteins [4] . Earlier, a complex of the RNA region and protein S4 (S4-RNP II) was characterised for (a) the identities of the nucleotide sequences that it contains, (b) the positions of the ribonuclease cuts which yield important topographical information about the RNA structure, and (c) the approximate localisation of those interacting regions that are distantly separated in the RNA sequence and facilitate the folding of the

Small-angle x-ray scattering study of S4-RNA, the 16 S RNA binding site for the protein S4 from Escherichia coli ribosomes

The ribonucleic acid (16 S RNA) of the 30 S Escherichia coli ribosomal subunit binds many of the proteins in this subunit, such as, for instance, S4, S7, S8, S15 and S20 [1,2]. The 16 S RNA binding site for the S4 protein, S4-RNA, can be isolated in pure form [3-91 ; and it is indicated that this binding site consists mainly of two regions of approximately 150 and 160 nucleotides, originating from the 5’ terminal third of the 16 S RNA molecule [7]. These two regions, which are separated by about 120 nucleotides of the 16 S RNA sequence, seem to be stabilized by a specific RNA-RNA interaction [7] . Although the sequence of the S4-RNA region, prepared with both T1 and pancreatic ribonucleases, has been partially analysed [7,9], the correct molecular weight and the size and shape of S4-RNA are not known. We have analysed S4-RNA by using the small-angle X-ray scattering method; the results yield a molecular weight of 136 000 and a radius of gyration of 43.5 A. The X-ray scattering curve can be explained, in its proximal angular range, by the scattering from a twoparameter, uniform electron density model with a shape of an oblate ellipsoid and the dimensions of 132 X 132 X 32 a. 2. Materials and methods

Identification of the clathrin assembly protein AP180 in crude calf brain extracts by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We present a two-dimensional gel electrophoretic method which affords a diagnostic means for the identification of the neuron-specific clathrin assembly protein AP180 in crude cytosolic and microsomal fractions of bovine brain. The method is based on the finding that in the presence of sodium dodecyl sulfate (SDS) in a newly developed continuous high salt Tris-acetate-EDTA buffer system protein AP180 migrates at a rate corresponding to its molecular weight of approximately 120,000, while in other more commonly used SDS-polyacrylamide gel electrophoresis methods it behaves anomalously as a 170- to 180-kDa polypeptide. By combining electrophoresis in the Tris-acetate-EDTA system in the first dimension with either the electrophoretic system of Laemmli [Laemmli, U.K. (1970) Nature (London) 227, 680-685] or that of Neville [Neville, D.M. (1971) J. Biol. Chem. 246, 6328-6334] in the second dimension, it is possible to identify AP180 in complex protein mixtures, because it is the only major protein that fell significantly off a diagonal defined by other proteins. A comparison of the microsomal and soluble fractions examined in this manner reveals that most of the AP180 is present in the soluble fraction.

Absence of interaction between the 165-kDa fibronectin-binding protein involved in mouse odontoblast differentiation and vinculin

Previous data suggested that matrix could control the organization of microfilaments in differentiating odontoblasts and that this process involved a complex of fibronectin-165-kDa membrane protein-vinculin. The use of two different gel systems and microsequence analysis demonstrated that two distinct 165-kDa proteins interact, one with fibronectin and the other with vinculin.

Native and denatured structures within the 5′-region of 16 S ribosomal RNA from Escherichia coli

Native and denatured forms of small RNA molecules have been well-characterised, especially for the 5 S RNA of Escherichia coli [ 1,2]. These RNA forms can be readily distinguished by their electrophoretic properties [2], and by their ribosomal protein binding capacities [3]. There have also been reports of different conformational forms of the larger bacterial ribosomal RNAs that can be separated electrophoretically. However, in most of these studies the RNA was examined in the presence of EDTA and no criterion for the nativity of a conformation was applied, such as the specific interaction with ribosomal proteins [4-71. Since it is known that magnesium ions are essential for the ‘native’ conformations of the RNA sites of ribosomal proteins [8,9], these results probably only reflect different denatured states of 16 S RNA. In the present work, we show that RNA extracted by a phenol-dodecylsulphate procedure contains a mixture of three conformational forms that can be resolved electrophoretically in the presence of magnesium. We have denoted them ‘native’ (N), intermediate (I) and denatured (D) forms of 16 S RNA. The ‘native’ form was distinguishable from the denatured form in that it contained the complex RNA tertiary structure in the 5’-half of 16 S RNA that constitutes the RNA binding site of protein S4

Assembly Proteins and Adaptors in Clathrin Coated Vesicles

Clathrin coated vesicles, which are present in probably all eukaryotes, are the agents for receptor mediated endocytosis and for the transport of lysosomal enzyme receptors from the trans-Golgi to the endosomal system (Goldstein et al 1985; Pearse and Bretscher, 1981). In neurons they are conjectured to facilitate also the recycling of synaptic vesicle membrane after neurotransmitter release (Heuser 1989). The functions of the coat components include the binding of specific cargo molecules (receptors), the coordinated assembly of the coat, its reorganization and eventually its dissolution. The emphasis in this report lies on structural and functional aspects of clathrin associated proteins present in coated vesicle populations from bovine brain.