Purification, in vitro reassembly, and preliminary sequence analysis of epiplasmins, the major constituent of the membrane skeleton of Paramecium.
The epiplasmic layer, a continuous rigid granulo-fibrillar sheet directly subtending the surface membranes of Paramecium, is one of the outermost of the various cytoskeletal networks that compose it cortex. We have previously shown that the epiplasm consists of a set of 30 to 50 protein bands on SDS-PAGE in the range 50 to 33 kDa, the epiplasmins. We report a purification procedure for the set of epiplasmic proteins, a description of their physicochemical and reassembly properties, and a preliminary characterization of their sequence. The conditions for solubilization of the epiplasm and for in vitro reassembly of its purified constituents ar described. Reassembly of the entire set of proteins and of some (but not all) subsets are shown to yield filamentous aggregates. Microsequences of two purified bands of epiplasmins reveal a striking amino acid sequence consisting of heptad repeats of only three main amino acids, P, V, and Q. These repeats were confirmed by DNA sequencing of polymerase chain reaction products. The motif is QPVQ-h, in which h is a hydrophobic residue. This may constitute the core of the epiplasmin sequence and, in view of the tendency of such a sequence to form a coiled-coil, may account for the remarkable self-aggregation properties of epiplasmins.
Coffe
G
G
Laboratoire de Biologie Cellulaire 4, URA 1134, Université de Paris-Sud, Orsay, France.
Le Caer
J P
JP
Lima
O
O
Adoutte
A
A
D000595:Amino Acid Sequence; D000818:Animals; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D016054:DNA, Protozoan / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016805:Paramecium tetraurelia / Q000737:chemistry* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D012995:Solubility; D014981:Xenopus
D003598:Cytoskeletal Proteins; D016054:DNA, Protozoan; D015800:Protozoan Proteins