Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally ( pf-18, pf-19, and pf-15) or nearly totally ( pf-20) the central microtubular pair complex in isolated axonemal preparations . Analysis of "S-labeled axonernal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes . Analyses of axonemal proteins labeled by cellular growth on 32 P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated .Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-a or anti-/3 tubulin sera prepared from Chlamydomonas tubulins . The immunoprecipitates identify two major a tubulins, a major # tubulin, and a minor component which is also precipitated by the anti-f3 serum . None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella .The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules . Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.The analysis of axonemal polypeptides in mutants of Chlamydomonas reinhardtii with defects in flagellar structures has proved to be a valuable approach to the study of assembly and function of eukaryotic flagella. Using electrophoretic methods which identify nearly two hundred axonemal polypeptides, wild-type axonemes have been compared with those from mutants lacking radial spokes (1, 2, 3) and inner or outer dynein arms (4) . In each case, the comparison revealed that the mutant axonemes were deficient in a set of polypeptides, each set being unique for the missing structure . The missing polypeptides identify possible structural components of the radial spokes or dynein arms; they also serve as a signature by THE JOURNAL OF CELL BIOLOGY " VOLUME 91 OCTOBER 1981 69-76 © The Rockefeller University Press -0021-9525/81/10/0069/08 $1 .00 which additional mutants affecting these structures can be identified or by which polypeptides present in the course of enzyme purification can be identified with specific axonemal structures . The usefulness of such signatures is exemplified by the identification of several mutants affecting radial spokes (2, 3) and by progress in purification of dyneins (4,5) .This paper is concerned with the flagellar and axonemal polypeptides of a series of mutants defective in the central microtubules and their appended struct...

Abstract. Modification of macrophage phagosomes begins shortly after formation as 7bxop/asma cells secrete membranous vesicles that form a reticulate network within the vacuole. The/bxop/asma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca ++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein.The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxop/asma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular/bxop/asma cells as shown by immunopemxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles.Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the/bxop/asma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-I flagella can be at virtually any location on the cell surface, vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.The basal body developmental cycle of the unicellular biflagellate green alga, Chlamydomonas reinhardtii, represents a clear case in which organelle replication is coupled to cell replication. In the basal body cycle, new basal bodies form at determined times and places during interphase, and they segregate equally at cell division to yield biflagellate daughter cells (2,8). Through the analysis of mutants with defects in the basal body cycle, we hope to gain insight into the mechanisms of basal body biogenesis and segregation, and also into the mechanisms by which cell and organelle cycles are coordinated. In this communication we describe a mutant, vfl-1, in which the temporal and spatial regulation of the basal body cycle is defective. As a result, vfl-1 cells have the novel property of assembling new flagella continuously throughout much of the cell cycle.

We have identified the fifth member of the mammalian X,K-ATPase L L-subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as L L muscle (L L m ) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of L L m proteins with known Na,K-and H,K-ATPase L L-subunits are 30.5^39.4%. Unlike other L L-subunits, putative L L m proteins have large N-terminal cytoplasmic domains containing long Glurich sequences. The data obtained indicate the existence of hitherto unknown X,K-ATPase (most probably Na,K-ATPase) isozymes in muscle cells.z 1999 Federation of European Biochemical Societies.

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