Multiple families of proteins in the secretory granules of Paramecium tetraurelia: immunological characterization and immunocytochemical localization of trichocyst proteins.
Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14-25 x 10(3)), acidic (pI 4.4-5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb A1-3 and B5-5) showed a relatively wide range of pI values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group II proteins (defined by mAb B4-4 and B3-5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electron-dense striations with 55 nm intervals. However, these regions were not accessible to antibody B4-4 in ctmx. Group III proteins (defined by mAb B7-4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups IV are two subunits of heterodimers. Proteins of groups III and IV were localized in the core of ctmx, but were distributed uniformly in xtmx. Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.
Shih
S J
SJ
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal; D003594:Cytoplasmic Granules / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016805:Paramecium tetraurelia / Q000737:chemistry*; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D015800:Protozoan Proteins