Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells.

Abstract

Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated Staphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent . The specificity of the immunoadsorption was monitored by SDS PAGE analysis and by competitive ELISA assays . SDS PAGE of the material immunoadsorbed from a fraction of porcine brain smooth microsomes showed a selective enrichment in a 180,000 mol wt protein. In an ELISA assay, this protein competed effectively-in binding anticlathrin-with clathrin extracted from a coated vesicle preparation . When the immunoadsorbed fraction was examined by electron microscopy, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles . Upon extensive dialysis (against MES buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin .This study demonstrates that anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles . In addition, the ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution .The ubiquitous presence of coated vesicles in eukaryotic cells has generated a great deal of interest and speculation concerning the role(s) these organelles play in cellular processes . Recent studies have shown that coated vesicles are involved in receptor-mediated endocytosis (2,4,14,15,17,38,40,43), formation of primary lysosomes (13), membrane recycling (5, 18, 19), and intracellular vesicular transport (11-13, 36, 41).The pertinent evidence has been derived either from morphological observations of cells actively engaged in one of the processes mentioned, or from cell fractionation studies . Both of these general approaches have technical limitations when applied to the study of coated vesicles because of the transient existence of these structures and the fragility of their clathrin coat. Besides, with these procedures, many important questions concerning the mechanisms involved in the assembly and/or disassembly of coated vesicles in situ, or the fate of the clathrin cage upon disassembly from the vesicle are virtually impossible to answer .In an attempt to improve on available isolation procedures and to solve some pending problems concerning the formation and function ofcoated vesicles, we have developed a technique that uses anticlathrin antibody for the affinity separation of coated vesicles from crude fractions of tissue homogenates . In this report, we show that formaldehyde-fixed, heat-inactivated Staphylococcus aureus cells can be used as a solid phase immunoadsorbent for the isolation of coated vesicles. With this procedure, it is possible to: (a) increase the ef...

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