Rapamycin is a potent immunosuppressant that blocks the G1/S transition in antigen-activated T cells and in yeast. The similar effects of rapamycin in animal cells and yeast suggest that the biochemical steps affected by rapamycin are conserved. Using a two-hybrid system we isolated mammalian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin. Specific interactors, designated RAPT1, encode overlapping sequences homologous to yeast Tor, a putative novel phosphatidylinositol 3-kinase. A region of 133 amino acids of RAPT1 is sufficient for binding to the FKBP12/rapamycin complex. The corresponding region in yeast Tor contains the serine residue that when mutated to arginine confers resistance to rapamycin. Introduction of this mutation into RAPT1 abolishes its interaction with the FKBP12/rapamycin complex.
Abstract. BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for ~-or/~-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over-or underexpression of BIKI causes aberrant microtubule assembly and function, bikl null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bikl cells.
A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.Asexual reproduction in Neurospora crassa involves the differentiation of three cell structures-mycelia or vegetative hyphae, aerial hyphae, and conidia, the asexual spores. N. crassa can be grown under conditions that either promote vegetative growth or induce conidiation. In liquid medium, under continuous agitation, only vegetative growth occurs. However, on a solid surface or when mycelia are harvested onto filter paper and incubated under aerobic conditions, the vegetative hyphae undergo a number of morphological changes, possibly in response to aerobiosis and desiccation or nutrient limitation. The abrupt change in growth conditions upon filtration triggers the steps leading to conidiation, which then proceed in a synchronous fashion. Within 2 h vegetative hyphae begin to grow upward, perpendicular to the surface of the substrate, and differentiate into aerial hyphae. The aerial hyphae elongate, branch, and by 12 h produce large numbers of conidia by repeated apical budding. By manipulating the growth conditions in this way, homogeneous populations of cells at different stages of differentiation can be readily isolated and analyzed (1).Studies of asexual differentiation in Aspergillus nidulans have shown that approximately 1,000 polyadenylated [poly(A)+] RNA sequences, representing 6% of the genome, preferentially accumulate in cultures induced to conidiate (27). Several lines of evidence suggest that differential gene expression also occurs during the formation of aerial hyphae and conidia in N. crassa. First, a number of "phase-specific" mutants have been isolated that are defective in the differentiation of aerial hyphae or conidia, but are unaffected in vegetative growth or sexual reproduction (13,22). Second, on two-dimensional gels of proteins synthesized during the asexual cycle, 12 polypeptides were detected in extracts of aerial hyphae or conidia that were not readily detected in extracts of...
The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in ceils blocked in N-glycosylation-i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion. The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2. UPR elements present in other ER chaperone genes, such as yeastKAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2. As with KAR2, FKB2 mRNA levels are also elevated by heat shock. The similarities in the regulation ofFKB2 and other ER the Drosophila melanogaster photoreceptor-specific cyclophilin homolog ninaA, which is required for transport of rhodopsin through the secretory pathway (9). Mutant alleles of ninaA define a region required for rhodopsin biogenesis which encompasses the peptidyl-prolyl substrate-binding site, indicating that this is the functionally relevant region of the molecule (10). The recent finding that a CsA-sensitive immunophilin can accelerate the rate of protein folding of carbonic anhydrase by PPIase-dependent and -independent mechanisms suggests that immunophilins may be multifunctional (11).Other immunophilins are found as constituents of protein complexes. FKBP-52 (12), also called FKBP-59 (13), p56 (14), and hsp56 (15), associates with the 90-kDa heat shock protein (hsp9O) in the inactive steroid receptor complex (16); FKBP-12 binds with high affinity to the Ca2+-release channel in skeletal muscle (17); and s-cyclophilin colocalizes with the Ca2+-storage protein calreticulin (18). These observations suggest a role for immunophilins in steroid receptor function and in calcium regulation. However, the functional significance of these protein-protein interactions has not been established.Yeast FKBPs, like their mammalian counterparts, possess PPIase activity that is inhibited by FKS06 and rapamycin (19)(20)(21) Since certain genes specifying cellular chaperones are regulated in response to stress (24-26), we considered that this might be the case for FKBP-13. We report here that FKB2 is regulated by two forms of stress: heat shock and conditions that cause accumulation of unglycosylated precursor proteins in the ER. The regulation of FKB2 by increased levels of precursors in the ER parallels that of other ER chaperone genes: yeast KAR2 (BiP), required for entry of proteins into the ER (27), and mammalian GRP78 (BiP) and GRP94 (26).Abbreviations: ER, endoplasmic reticulum; UPR, unfolded-protein response; FKBP, ...
The asexual developmental pathway in the life cycle of the filamentous fungus Neurospora crassa culminates in the formation of spores called conidia. Several clones of genomic Neurospora DNA have been isolated that correspond to mRNA species expressed during conidiation and not during mycelial growth (V. Berlin and C. Yanofsky, Mol. Cell. Biol. 5:849-855, 1985). In this paper we describe the characterization of one of these clones, named pCon-lOa. This clone contains two genes, con-10 and con-13, which are induced coordinately during the later stages of conidiation. The two genes are separated by 1.4 kilobases of DNA; they are located on linkage group IV and are transcribed from the same strand of DNA. The molecular organization and sequence of one of these genes, con-10, and its flanking regions are presented. Full-length cDNA clones for con-10 also were isolated and sequenced, and transcription-initiation and polyadenylation sites were defined. The con-10 gene contains an open reading frame interrupted by two small introns and encodes an 86-amino-acid residue polypeptide that is both hydrophilic and weakly acidic. Expression of the con-10 gene in various mutants defective at different stages of conidiation indicates that it plays a role after aerial hyphal development. Possible functions, organization, and regulation of conidiation-specific genes are discussed.Under vegetative conditions, Neirospora crassa grows as a mycelium composed of branching hyphae. During the asexual phase of its life cycle, aerial hyphae develop perpendicularly from the mycelium. Asexual spores called macroconidia develop at the distal ends of branches of these aerial hyphae (49; macroconidia will be called conidia throughout this paper). The environmental and physiological factors that trigger this process are unclear but are thought to involve dessication and nutrient deprivation (40). Microscopic studies have shown that developmental stages in the conidial pathway are fairly ordered (37). Since major changes in gene expression occur during the asexual cycle of N. crassa, as shown by alterations in steady-state levels of many proteins and mRNAs (7,55), it is likely that conidiation involves selective expression of subsets of this organism's genes. Some changes associated with conidial development appear to involve shifts in metabolic functions (12), whereas others lead to the synthesis of novel structural components (4). Genetic studies with N. crassa have identified many loci involved in conidiation (37,44,50). Mutations in these genes result in an altered morphology of the culture attempting conidiation or prevent conidiation, but little is known about the lesions responsible for these phenotypes. In the related filamentous fungus Aspergillius niidiulans, three genes that cause defective conidial development have recently been cloned (10,27). Initial characterization has shown that these genes are transcribed only during conidiation.We would like to determine the regulatory events that mediate the process of conidiation. Our appro...
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