Rapid purification of clathrin-coated vesicles by free-flow electrophoresis

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.     

Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane

Clathrin in coated vesicles is linked to transmembrane receptors by adaptor protein complexes. The Golgi-associated adaptor complex HA1 is a tetramer, made up of beta', gamma, 47-kDa, and 20-kDa subunits, whereas the tetrameric plasma membrane adaptor, HA2, contains alpha, beta, 50-kDa, and 16-kDa subunits (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929). Here we report on the structural organization of adaptor subunits as revealed by proteolytic dissection. We show that the beta' and gamma subunits of HA1 are cleaved into 60-67-kDa "trunk" and 32-44-kDa "head" fragments. Interactions between adaptor subunits involve the trunk domains only. In overall organization of their domains, the Golgi and plasma membrane adaptors are very similar. The similarity encompasses also the location of phosphorylated serine residues in the alpha a, beta, beta', and gamma subunits, which are found in the head domains in all cases. In the alpha a and beta subunits they probably occur in the proline- and glycine-rich hinge region, which connects the head to the trunk. Identical adaptor fragments were obtained by controlled digestion of clathrin-coated vesicles. Under conditions that did not affect the integrity of the clathrin heavy chain, the adaptor head fragments were always quantitatively released from coated vesicles. The release of the bulk of the adaptors occurred concomitantly with the cleavage of their beta-type subunits (beta and beta') and under buffer conditions that prevent aggregation of adaptors. These observations taken together with the results of reconstitution experiments confirm and extend previous data (Ahle, S., and Ungewickell, E. (1989) J. Biol. Chem. 264, 20089-20093) which suggested that adaptors attach to clathrin through their beta-type (beta and beta') subunits. Moreover, high affinity interaction between adaptors and clathrin requires the participation of regions from both the head and trunk domains of the beta-type subunits.     

Evidence for tertiary structural RNA-RNA interactions within the protein S4 binding site at the 5''-end of 16S ribosomal RNA of Escherichia coli.+.

Evidence is presented for tertiary structural interaction(s) (interactions(s) between two regions of an RNA molecule that are widely separated in the RNA sequence) within the 5'-one third of the 16S ribosomal RNA of Escherichia coli that constitutes the binding site of protein S4. The two main interacting RNA regions were separated by about 120 nucleotides (sections Q to M) of the 16S RNA sequence. A second, smaller gap, of 13 nucleotides, occurred within section C". The two main interacting regions contain about 150 nucleotides (sections H" to Q) and 160 nucleotides (sections M to C"). They are folded back on one another and, especially in the presence of protein S4, are strongly protected against ribonuclease digestion. The intermediate region (sections Q to M), however, is relatively accessible to ribonucleases in the S4-RNP. By partial removal of subfragments from the RNA complex it was possible to localise the two main interacting sites within sections H" - H and sections I" - C". Three main criteria for the specificity of the RNA-RNA interactions were invoked and satisfied. The possibility of other tertiary structural RNA-RNA interactions occurring in other regions of the 16S RNA is discussed. Finally, all the structural information on the S4-RNP is summarised and a tentative model is proposed.     

Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.     

Endocytosis: clathrin-mediated membrane budding

Clathrin-dependent endocytosis is the major pathway for the uptake of nutrients and signaling molecules in higher eukaryotic cells. The long-held tenet that clathrin-coated vesicles are created from flat coated plasma membrane patches by a sequential process of invagination, bud formation and fission recently received strong support from the results of advanced live cell fluorescence microscopy. The data on the critical components that deform the plasma membrane locally into a coated bud suggest that membrane bending is a team effort requiring membrane-curving protein domains, actin dynamics and, last but not least, clathrin. The scission step requires the mechano-enzymatic function of dynamin, actin dynamics and possibly myosin motor proteins. Finally, a burst of auxilin/GAK initiates the uncoating of the vesicle.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.