Abstract. Immature secretory granules (ISGs) in endocrine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lonvard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82: 5385-5389; Tooze, J., and S.A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained an enigma. Here we demonstrate using three independent techniques that immature secretory granules isolated from the rat neuroendocrine cell line PC12 have clathrin coat components associated with their membrane. To study the nature of the coat association we have developed an assay whereby the binding of the AP-1 subunit ~/-adaptin to ISGs was reconstituted by addition of rat or bovine brain cytosol. The amount of 7-adaptin bound to the ISGs was ATP independent and was increased fourfold by the addition of GTPTS. The level of exogenous ~/-adaptin recruited to the ISG was similiar to the level of 7-adaptin present on the ISG after isolation. Addition of myristoylated ARF1 peptide stimulated binding. Reconstitution of the assay using AP-1 adaptor complex and recombinant ARF1 provided further evidence that ARF is involved in 7-adaptin binding to ISGs; BFA inhibited this binding. Trypsin treatment and Trisstripping of the ISGs suggest that additional soluble and membrane-associated components are required for 7-adaptin binding.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x L protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x L . Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.Programmed cell death (PCD) in metazoans is an essential homeostatic mechanism permitting the removal of surplus cells during morphogenesis and tissue maintenance and the deletion of cells that present a risk to the organism because they are mutated or infected (10,19,35,36,40). For vertebrates, the descriptive name commonly given to the process of PCD is apoptosis. Classical apoptosis is characterized by membrane blebbing, cell shrinkage, chromatin condensation, and nuclear and cellular fragmentation, and it results from the activation of an intrinsic suicide program (47). Recent studies implicate the dysregulation of PCD in the pathophysiology of several human diseases, including AIDS (12, 28), neurodegenerative disease (25,37,44), and cancer (for a review, see reference 42).The basal machinery responsible for metazoan PCD is highly evolutionarily conserved and, at its execution level, involves the action of a discrete class of cysteine proteases, of which the prototypes are the interleukin-1␤-converting enzyme in humans and Ced-3 in the nematode (49). Also conserved are key regulators of apoptosis: in Caenorhabditis elegans the Ced-9 protein and in humans the Bcl-2 protein family (18, 46), which comprises both suppressors (e.g., Bcl-2 and Bcl-x L ) and promoters (e.g., Bax and Bak) of PCD (17,30,46).Recently, there have been several reports describing apparent PCD in the unicellular eukaryotes Tetrahymena thermophila, Dictyostelium discoideum, Trypanosoma brucei rhodsiense, and Trypanosoma cruzi and even in bacteria (2, 6, 45, 48; for an overview, see reference 1). PCD in unicellular organisms might facilitate constant selection for the fittest cell in the colony or optimal adaptation of cell numbers to the environment or might serve as a means for altruistic cell death to prevent the spread of virus in the event of infection.It has been shown that expression of the mammalian Bax protein in th...

Eukaryotic cells encode two homologs of Escherichia coliRecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

No abstract

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.