Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH 2 -terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER-and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [ Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed. † These authors contributed equally to this work. INTRODUCTIONProtein transport through the secretory pathway involves multiple steps that are conserved throughout eukaryotes (Palade, 1975;Schekman, 1985;Rothman, 1994;Schekman and Orci, 1996). Secreted or cell surface proteins are first translocated into the endoplasmic reticulum (ER) and then packaged into COPIIcoated vesicle intermediates for transport to the Golgi complex. Upon reaching the trans-Golgi, these proteins undergo another vesicle packaging event and enter secretory vesicles that ultimately dock and fuse with the plasma membrane. A striking and readily observable example of the secretory process is that of polarized secretion in the budding yeast Saccharomyces cerevisiae, where efficient targeting of secretory vesicles to newly em...

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.When DNA is damaged or chromosomes are incompletely replicated, cells become checkpoint arrested. These checkpoints avoid replication of damaged template DNA and prevent aberrant segregation of damaged or partly replicated chromosomes. In budding yeast, proteolysis of anaphase inhibitors is regulated by these checkpoint systems. Progression from metaphase to anaphase is inhibited by Pds1p in Saccharomyces cerevisiae (6,7,29,30). Before anaphase, Pds1p binds to Esp1p, inhibiting its anaphase-promoting activity (3). During an unperturbed cell cycle, Pds1p becomes polyubiquitinated at the metaphase-to-anaphase transition by multienzyme anaphase-promoting complex (APC)-cyclosome complexes. The modified forms are then recognized and degraded by 26S proteasomes (7). Once released from Pds1p, Esp1p activity induces the onset of anaphase.pds1 mutants fail to execute checkpoint control in response to DNA damage, spindle poisons, or replication inhibition (4, 29, 30). Pds1p is required for replication checkpoint control only late in S phase, not in the context of an early S-phase replication block enforced by hydroxyurea (HU) (4,29,30). In the presence of 0.1 M HU, replication proceeds more slowly. Under these conditions, cells perform other aspects of cell cycle progression, budding, and spindle assembly as rapidly as in the absenc...

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.

Abstract. The ellipsoidal shape of the yeast Saccharomyces cerevisiae is the result of successive isotropic/apical growth switches that are regulated in a cell cycledependent manner. It is thought that growth polarity is governed by the remodeling of the actin cytoskeleton that is itself under the control of the cell cycle machinery. The cell cycle and the morphogenesis cycle are tightly coupled and it has been recently suggested that a morphogenesis/polarity checkpoint control monitors bud emergence in order to maintain the coupling of these two events (Lew, D. J., and S. I. Reed. 1995. J. Cell BioL 129:739-749). During a screen based on the inability of cells impaired in the budding process to survive when the morphogenesis checkpoint control is abolished, we identified and characterized BED1, a new gene that is required for efficient budding. Cells carrying a disrupted allele of BED1 no longer have the wild-type ellipsoidal shape characteristic of S. cerevisiae, are larger than wild-type cells, are deficient in bud emergence, and depend upon an intact morphogenesis checkpoint control to survive. These cells show defects in polarized growth despite the fact that the actin cytoskeleton appears normal. Our results suggest that Bedl is a type II membrane protein localized in the endoplasmic reticulum. BED1 is significantly homologous to gma12 ÷, a S. pombe gene coding for an et-l,2-galactosyltransferase, suggesting that glycosylation of specific proteins or lipids could be important for signaling in the switch to polarized growth and in bud emergence.T HE ellipsoidal shape of the yeast Saccharomyces cerevisiae reflects cell cycle-regulated polarized growth. At specific times during the cell cycle, cell growth is either isotropic or polarized toward the bud (for review see Lew and Reed, 1995b). A correlation between local deposition of new cell wall components and actin localization has been established (Adams and Pringle, 1984;Kilmartin and Adams, 1984), leading to the proposal that actin directs secretory vesicles to specific regions of the plasma membrane to allow localized cell surface growth during bud initiation and bud growth. During most of the G1 phase, growth is isotropic and cortical actin patches are delocalized throughout the cell. The attainment of a critical cell size and concomitant execution of START lead to the formation of an actin ring at the pre-bud site and the orientation of actin filaments toward this site. Subsequent to START, growth is almost completely restricted to the emerging bud. During bud growth, cortical actin patches are localized to the bud. Initially, bud growth occurs primarily at the distal tip. At some point, though, there is a switch to isotropic growth first in the bud, then also tranAddress all correspondence to S. I. Reed, Department of Molecular Biology, MB7, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California 92037. Tel.: (619) 554-6188. siently in the mother cell at mitosis. At cytokinesis, the actin cytoskeleton is reorganized and...

During lytic infection, the adenovirus major late promoter (MLP) is primarily activated after the onset of viral DNA replication. Using a combination of DNA binding and in vitro transcription assays, we delineated a discrete MLP element spanning positions +80 to +106 which is essential for the replication-dependent activation of this promoter. We also identified a 40-kilodalton protein (the downstream element factor [DEF]) which binds to the +86-TTGTCAGTTT-+95 motif within this element. Whereas the DEF-binding activity is barely detectable in uninfected cells, it is readily visualized in adenovirus-infected cells, but only after the onset of viral DNA replication. Preventing the interaction of DEF with the MLP template impairs the in vitro transcriptional stimulation. We conclude that this replication-dependent activation of the MLP is, at least in part, mediated by induction of the specific binding of DEF to the MLP downstream element. * Corresponding author. MLP, that is essential for efficient transcription initiation from this promoter in replicating virus or plasmid constructs. Their results clearly show that a transacting factor(s) encoded or induced by adenovirus is required in addition to DNA replication. Using a cell-free transcription system initially developed by Leong and Berk (25) and based on extracts from cells infected with the wild-type adenovirus type 5 (wt) or its Ela-defective d1312 derivative (dl), we (21) recently confirmed that sequences upstream of +33 were involved in the Ela responsiveness of the MLP in vitro and demonstrated that sequences between +33 and +131 were implicated in the replication-induced activation of the MLP. In the present report, we further extend this study and show that the efficient binding of a 40-kilodalton (kDa) protein (downstream element factor [DEF]) to an element located between +80 and +106 is responsible for the transcriptional stimulation of MLP observed in wt-infected cell extracts compared with dl-infected cell extracts. Our results indicated that induction of this DEF-binding activity depends on viral DNA replication before extract preparation and suggested that the Ela gene products are dispensable for at least part of this induction. MATERIALS AND METHODS Preparation of whole-cell extracts. HeLa cells grown in Eagle medium supplemented with 5% calf serum were infected with 10 PFU of adenovirus type 5 (wt) or its Eladefective dl312 derivative (dl) per cell. For replicationblocked extracts, cytosine arabinoside (araC) was added at a final concentration of 20 ,ug/ml to the growth medium right after adsorption of the virus and 12 h later (25). Extracts were prepared 20 or 48 h postinfection (p.i.) by the method of Manley et al. (30), but the final dialysis was against buffer A (21) containing 50 mM Tris hydrochloride (pH 7.9), 12.5 mM MgCl2, 40 mM ammonium sulfate, 0.1 mM EDTA, 2 mM dithiothreitol, and 17% glycerol. Protein concentration was adjusted to 6 ,ug/,ul after dialysis. Partial purification of DEF. A whole-cell extract (70 ml, 420 mg of protein) ...

Transcription from the adenovirus major late promoter (MLP) is greatly stimulated during lytic infection, after replication of the viral DNA has started. This replication-dependent activation has previously been shown to be mediated by a positive regulatory cellular protein(s). Binding of this factor(s) to sequence elements (DE1 and DE2), located between positions +76 and +124, with respect to the MLP transcriptional startsite, is detected only after the onset of DNA replication. Using a cell-free transcription system which mimics the late phase induction of the MLP and DNA binding assays, we now present evidence showing that maximal stimulation also depends on the MLP upstream element (UE), without involving increased DNA binding activity of the corresponding factor (UEF) during the lytic cycle. Our results indicate that the upstream and downstream elements act cooperatively on transcription efficiency, although no direct interactions between the cognate factors could be demonstrated. These observations strongly suggest that the elevated rate of transcription originating at the MLP startsite, late in infection, results from the simultaneous action of factors bound at the upstream and downstream elements onto a common target within the basal transcription machinery.