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The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19’s localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.

Das Verbundverhalten von Betonschrauben mit grosem Durchmesser wird mittels Auszugsversuchen mit enger Stutzung untersucht. Durch das Eindrehen von Betonschrauben in das erstellte Bohrloch wird eine kraftschlussige Verbindung hergestellt, uber deren Verhalten wenig bekannt ist. Zusatzlich zur konventionellen Installation auf Basis des Hinterschnitts wurde eine Variante mit zusatzlichem Verbundmortel untersucht. In ersten Versuchen wird der Einfluss der Verbundlange der Schrauben auf das Tragverhalten durch Variation der Anzahl an Gewindegangen fur verschiedene Betondruckfestigkeiten betrachtet. Um genauere Auskunft uber die Spannungsverteilung im Kraftubertragungsbereich zu erhalten, wurden anschliesend zwei Arten von Messschrauben mit Dehnmessstreifen im Verbundbereich entwickelt. Aus den Erkenntnissen, die mit diesen Messschrauben gewonnen wurden, wird eine Ansatzfunktion abgeleitet, mit welcher die Verteilung der Langsspannung beschrieben werden kann. Aus dieser Verteilung kann anschliesend analytisch auf die Verbundspannungsverteilung ruckgerechnet werden. Abschliesend wird ein Vergleich mit dem gangigen Ansatz einer konstanten Verbundspannung untersucht.

The Altstadtring-Tunnel is one of the essential east-west traffic routes in the city center of Munich and was constructed in the late 1960s. Segment 34 of the tunnel was built directly underneath the existing Prince-Carl- Palais, a historic building from 1804. Therefore 15 pre-stressed concrete girders with an effective depth of3.5 m and a maximum span of up to 30 m were built which now form the tunnel roof slab. These girders were pre-stressed with steel nowadays well known for stress corrosion cracking. A recalculation of the slab showed that no ductile failure can be guaranteed in case of a progressive rupture of the tendons. Therefore, a concept for strengthening the slab was developed using concrete screw anchors as post installed bending and shear reinforcement. The concrete screw anchors are normally installed as anchoring elements in cracked and non- cracked concrete and are available with diameters up to 22 mm. Developing this concept further, it is straight forward to use these anchoring elements as post-installed reinforcement in existing concrete structures. This new strengthening system was developed at the University of Innsbruck in the last few years and can fulfill the special requirements of this project, such as installation of the strengthening system from underneath the tunnel slab during ongoing use of the structure. High strength steel with diameters of up to 63.5 mm will be used as post-installed bending reinforcement covered with a new shotcrete layer on the underside of the tunnel slab. In total 59.3 tons of new flexural reinforcement and 7199 concrete screws for strengthening the shear capacity of the girders will be used to ensure a ductile failure of the tunnel slab. The on-site work started in March 2019 and is expected to take two years to complete.

Kinetochores that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. We show that they also promote their own capturing. Similar to what governs the localization of spindle assembly checkpoint proteins, the phosphorylation of Spc105 by Mps1 allows unattached kinetochores to sequester Stu1 in cooperation with Slk19. The withdrawal of Stu1, a CLASP essential for spindle integrity, from microtubules and attached kinetochores disrupts the organization of the spindle and thus allows the enhanced formation of dynamic random microtubules that span the nucleus and are ideal to capture unattached kinetochores. The enhanced formation of nuclear random microtubules does not occur if Stu1 sequestering to unattached kinetochores fails and the spindle remains uncompromised. Consequently, these cells exhibit a severely decreased capturing efficiency. After the capturing event, Stu1 is relocated to the capturing microtubule and prevents precocious microtubule depolymerization as long as kinetochores are laterally or incompletely end-on attached., Kinetochores (KT) that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. Here the authors show that Mps1 localizes Stu1 at unattached KTs together with Slk19, causing a reorganization of the nuclear MT network that favors the capturing of unattached KT.

Kurzfassung Die Industrie 4.0 zielt auf eine stärkere Vernetzung von Produktions- und Logistikprozessen ab. Dafür werden durchgängige Konzepte zur automatischen Identifizierung von Systemelementen benötigt. In diesem Beitrag wird daher ein Vorgehen für die Erstellung von geeigneten AutoID-Konzepten zur Steigerung der Flexibilität und Effizienz der Prozesse vorgeschlagen. Die Vorgehensweise zur Konzepterstellung wird anhand eines cloudbasierten Werkzeugmanagementsystems verdeutlicht.

Systems and applications within the industry 4.0 have a high demand for accurate and up-to-date information or context about their environment. A fundamental type of information is the identity of objects. In future systems, there is also an increased need for context on the location and the status of identified objects. RFID can already address the demand for location information by signal processing. In this paper, we describe a smart RFID reader that is furthermore able to collect and process context on logistic objects by including logistic data sources. The enhanced usage of context allows for an adaptable system that is able to analyze its measurements to recognize and report logistic process events. In order to obtain this functionality we extract the spatial structure of operation environments from 2D-CAD layouts of the warehouse. For the detection of process events, we developed a method to validate the measurements by reasoning. To obtain basic rules for the reasoning, we describe an ontology on basic interactions between logistic objects. The basic rules are then synchronized with the data form a warehouse management system by processing a form of decision trees.

The present publication describes a localization concept to determine the 3D Position of passive UHF-RFID transponders. The importance of accurate location information significantly increases with Industry 4.0 in mind. Various applications in production and logistics require the exact location for monitoring the processes and thereby increasing the quality and reliability. The presented system consists of an 8 channel superheterodyne receiver with a connected 2D L-shaped patch antenna array controlled by a standards-compliant UHF reader. The recorded backscattered signal from the transponder gets evaluated by 2D versions of Bartlett Beamforming and MUSIC algorithm using a calculated 2D array manifold. For distance estimation the phase of the backscattered transponder signal gets evaluated for different reader transmission frequencies in the UHF band by applying a modified MUSIC algorithm. By measuring the distance, azimuth and elevation angle a monostatic 3D localization of the passive transponder is possible. For validation the localization concept is examined under ideal conditions in an anechoic chamber and in an industrial environment to show the overall performance.

The two different TOGL domains of the budding yeast CLASP Stu1 are responsible for its distinct mitotic activities, and these activities are only partially mediated by tight microtubule binding., Cytoplasmic linker–associated proteins (CLASPs) are proposed to function in cell division based on their ability to bind tubulin via arrayed tumor overexpressed gene (TOG)–like (TOGL) domains. Structure predictions suggest that CLASPs have at least two TOGL domains. We show that only TOGL2 of Saccharomyces cerevisiae CLASP Stu1 binds to tubulin and is required for polymerization of spindle microtubules (MTs) in vivo. In contrast, TOGL1 recruits Stu1 to kinetochores (KTs), where it is essential for the stability and tension-dependent regulation of KT MTs. Stu1 is also recruited to spindle MTs by different mechanisms depending on the mitotic phase: in metaphase, Stu1 binds directly to the MT lattice, whereas in anaphase, it is localized indirectly to the spindle midzone. In both phases, the activity of TOGL2 is essential for interpolar MT stability, whereas TOGL1 is not involved. Thus, the two TOGL domains of yeast CLASP have different activities and execute distinct mitotic functions.

Localization of UHF RFID tags in an industrial environments is difficult due to signal reflections and multipaths caused by steel and metal objects. Existing solutions have shown decent accuracy for small distances but fail to maintain the accuracy as the distance between the antenna and the tag increases. In this paper, we describe a novel UHF RFID localization approach based on location fingerprinting. The approach uses machine learning to transform localization into a classification problem. Location fingerprints are generated using outputs Bartlett beamformer and MUSIC algorithms that estimate the incoming angle of a signal. We evaluated our approach in an industrial environment, and the results show that we achieve a high classification accuracy and maintain it with the increase of the distance between the tag and the antenna.

Impacts of the operation environment on the read field of passive UHF RFID applications can be decisive for the successful implementation of the systems. However, these impacts cannot easily be foreseen or detected. Therefore, it is necessary to verify the configuration of the installation regarding a reliable performance when putting the installation into operation. But the testing of a certain configuration can be a very complex and time consuming procedure, especially when approaching by trial and error. To support the evaluation of an installation's configuration and the detection of problematic areas within the read field, a new concept for a measurement tool is presented in this paper. The basic idea of the tool is to measure the spatial distribution of the field strength by tracking the measuring antenna's positions. For a quick understanding and analysis of the measurement's results, a 3D visualization is used. To evaluate the measurement tool, we conducted comparative measurements aimed at the detection of the weak areas of a read field at an RFID gate.

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

The Saccharomyces cerevisiae CLASP (CLIP-associated protein) Stu1 is essential for the establishment and maintenance of the mitotic spindle. Furthermore, Stu1 localizes to kinetochores. Here we show that, in prometaphase, Stu1 assembles in an Ndc80-dependent manner exclusively at kinetochores that are not attached to microtubules. Stu1 relocates to microtubules when a captured kinetochore reaches a spindle pole. This relocation does not depend on kinetochore biorientation, but requires a functional DASH complex. Stu1 at detached kinetochores facilitates kinetochore capturing. Furthermore, since most of the nuclear Stu1 is sequestered by one or a few detached kinetochores, the presence of detached kinetochores prevents Stu1 from localizing the spindle, and therefore from stabilizing the spindle. Thus, the sequestering of Stu1 by detached kinetochores serves as a checkpoint that keeps spindle poles in close proximity until all kinetochores are captured. This is likely to facilitate kinetochore biorientation. In agreement with the findings described above, a kinetochore mutant (okp1-52) that fails to release Stu1 from the kinetochore displays a severe spindle defect upon spindle pole body separation, and this defect can be rescued by destroying the okp1-52 kinetochore.

The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1–Bub2 guanosine triphosphatase–activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil–like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.

A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins, which have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. Here, we show that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore–microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains.

We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore. When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids. In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity. The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex. However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does. Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity. Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity.

CA125 is an ovarian cancer antigen whose recently elucidated primary structure suggests that CA125 is a giant mucin-like glycoprotein present on the cell surface of tumor cells. Here, we establish a functional link between CA125 and β-galactoside-binding, cell-surface lectins, which are components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression. On the basis of mass spectrometry and immunological analyses, we find that CA125 is a counter receptor for galectin-1, as both soluble and membrane-associated fragments of CA125 derived from HeLa cell lysates are shown to bind specifically to human galectin-1 with high efficiency. This interaction is demonstrated (1) to depend on β-galactose-terminated, O-linked oligosaccharide chains of CA125, (2) to be preferential for galectin-1 versus galectin-3 and (3) to be regulated by the cellular background in which CA125 is expressed. Despite lacking a conventional signal peptide, a CA125 C-terminal fragment of 1148 amino acids, representing less than 10% of the full-length protein, retains the ability to integrate into secretory membranes such as the endoplasmic reticulum (ER) and the Golgi, and is targeted to the plasma membrane by conventional secretory transport. As demonstrated by a novel assay that reconstitutes non-conventional secretion of galectin-1 based on fluorescence-activated cell sorting (FACS), we find that tumor-derived HeLa cells expressing endogenous CA125 present more than ten times as much galectin-1 on their surface compared with non-tumor-derived, CA125-deficient CHO cells. Intriguingly, both the galectin-1 expression level and the cell-surface binding capacity for galectin-1 are shown to be similar in CHO and HeLa cells, suggesting that CA125 might be a factor involved in the regulation of galectin-1 export to the cell surface.

Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs. Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p. In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation. In addition, Sac3p binds to the mRNA exporter Mex67p–Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC). Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects. Taken together, our data suggest that the novel Sac3p–Thp1p complex functions by docking the mRNP to specific nucleoporins at the nuclear entrance of the NPC.

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro . Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.

We show here that Ask1p, Dad2p, Spc19p and Spc34p are subunits of the budding yeast Duo1p-Dam1p- Dad1p complex, which associate with kinetochores and localize along metaphase and anaphase spindles. Analysis of spc34-3 cells revealed three novel functions of the Duo1-Dam1p-Dad1p subunit Spc34p. First, SPC34 is required to establish biorientation of sister kinetochores. Secondly, SPC34 is essential to maintain biorientation. Thirdly, SPC34 is necessary to maintain an anaphase spindle independently of chromosome segregation. Moreover, we show that in spc34-3 cells, sister centromeres preferentially associate with the pre-existing, old spindle pole body (SPB). A similar preferential attachment of sister centromeres to the old SPB occurs in cells depleted of the cohesin Scc1p, a protein with a known role in facilitating biorientation. Thus, the two SPBs are not equally active in early S phase. We suggest that not only in spc34-3 and Deltascc1 cells but also in wild-type cells, sister centromeres bind after replication preferentially to microtubules organized by the old SPB. Monopolar attached sister centromeres are resolved to bipolar attachment in wild-type cells but persist in spc34-3 cells.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.

We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins—Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1—as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein–DNA conformation that evokes the active centromere.

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N-terminal domain of Ndc80 (Ndc80(1-257)), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc80(1-257) in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild-type-like bipolar kinetochore-microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1-dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores.

The cell wall of fungi is a highly complex structure consisting of a network of polysaccharides ill which a plethora of different proteins are embedded. It is one of the major organelles of the cell surrounding it like an armor which protects from environmental stresses like osmotic pressure and defines the shape and physical strength or the fungal cell. It is crucial for colonization and infection since it defines the interface between host and pathogen. No similar structure is present in the host, therefore it defines a prime target for drug development. In this context, it has been shown that cell surface proteins are required for adhesion to host cells. The fact, that both pathogenic fungi, like Candida albicans as well as non-pathogenic fungi.. like Saccharomyces cerevisiae, in general, have a very similar polysaccharide structure but differ significantly in their protein composition which underscores the importance of cell wall proteins for pathogenesis. However, cell wall proteomics of fungi is a highly challenging task due to the complex biochemistry of these proteins. The extensive post-translational modifications and covalent attachment to the polysaccharide backbone of a large proportion of cell wall proteins makes it a demanding task to isolate and identify them. Ill this article, we describe the recent approaches that have been developed to describe cell wall dynamics and to isolate and identify cell wall proteins in the pathogenic yeast C. albicans.

12 páginas, 7 figuras, 1 tabla. Material suplementario en: https://doi.org/10.1016/S0092-8674(03)01025-0. The SUS1 sequences have been deposited in GenBank with the accession number AY278445., Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery, This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 352, Leibniz-Programm) and Fonds der Chemischen Industrie, R.R. by a grant from NIH, and J.E.P.-O. was supported by (GEN2001-4707-C08-07) from Ministerio de Ciencia y Tecnologı́a and (QLRI-CT-1999-01333) from the European Union. S.R.-N. is a holder of a Marie Curie fellowship.

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A(0), A(1), and A(2). Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

The centromere–kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein–DNA and protein–protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S -transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro . These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Yeast centromeric DNA ( CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3– CEN DNA complex by atomic force microscopy. Assembly of CBF3– CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.

A multisubunit protein complex, Cbf3, is a component of the Saccharomyces cerevisiae kinetochore. Cbf3 was recently shown to be essential for chromosome segregation in vivo and for movement of centromere DNA (CEN) along microtubules in vitro. Cbf3 contains three proteins, Cbf3a, Cbf3b and Cbf3c. Here the characterization of Cbf3b is described. Cbf3b contains an N-terminal Zn2Cys6 type zinc finger domain, a C-terminal acidic domain and a putative coiled coil dimerization domain. Cbf3b is essential for growth. Mutations within the zinc finger domain result in cells that exhibit a G2-M cell cycle delay and increased chromosome loss in each mitotic cell division. Therefore, Cbf3b has an essential function in chromosome segregation and the zinc finger domain executes part of this function presumably by providing the specific interaction between Cbf3 and CEN. Finally, data are provided to show that Cbf3c is encoded by CTF13, a gene that had been cloned recently by complementing a temperature sensitive mutant that exhibits chromosome loss as a result of a defective centromere.

A key protein component (CBF3) of the budding yeast (S. cerevisiae) centromere/kinetochore has been purified and characterized. CBF3 is a 240 kd multisubunit protein complex that binds specifically to the yeast wild-type centromere DNA (CEN), but not to nonfunctional CEN DNA containing a single base substitution in the critical CDEIII consensus sequence. When purified by affinity chromatography, CBF3 contains three protein components: CBF3A (110 kd), CBF3B (64 kd), and CBF3C (58 kd). Highly purified CBF3 requires the presence of a separate assembly factor or chaperone activity to bind to CEN DNA. Treatment with phosphatase inactivates CBF3, indicating that at least one of the CBF3 subunits must be phosphorylated for DNA binding to occur. A 56 bp region including the 26 bp CDEIII consensus is protected from DNAase I cleavage in the CBF3-CEN DNA complex.

The hexagonally patterned surface layer of halobacteria consists of a true glycoprotein. This procaryotic glycoprotein has recently been shown to exhibit novel features with respect to saccharide structure and saccharide biosynthesis. The primary structure and the location of glycosylation sites were determined by cloning and sequencing of the glycoprotein gene of Halobacterium halobium. According to the predicted amino acid sequence, the glycoprotein is synthesized with a N-terminal leader sequence of 34 amino acid residues reminiscent of eucaryotic and procaryotic signal peptides. A hydrophobic stretch of 21 amino acid residues at the C terminus probably serves as a transmembrane domain. 14 threonine residues are clustered adjacent to this membrane anchor and linked to these threonines are all the disaccharides of the cell surface glycoprotein. 12 N-glycosylation sites are distributed over the polypeptide chain.

In [l] we reported that the halobacterial cell wall glycoprotein, studied in detail [2], contains at least two species of sulphated saccharides: amino-sugar free oligosaccharides of low M, and one high M, saccharide (HM,-saccharide) containing mainly amino-sugars and galactose (heterosaccharide). The gel electrophoretic pattern of this sulphated ‘heterosaccharide’ gave rise to the assumption that it represents a repetitive sequence of a sulphated unit rather than a heterosaccharide structure. The function of this glycoconjugate in determining the rod form of the bacteria has been suggested [ 1,3]. Here we prove the suggested repetitive structure of this saccharide. Furthermore, a sulphated lipid-linked precursor of this glycoconjugate is described indicating a biosynthetic pathway different from that known for glycosaminoglycans.

Accurate chromosome segregation is DApenDAnt on a specialized chromosomal structure, the kinetochore/centromere. The only essential constituent of the S. cerevisiae kinetochore established today is CBF3, a multisubunit complex that binds to S. cerevisiae centromere DNA. Therefore CBF3 and its four components, Cbf3a, Cbf3b, Cbf3c and Cbf3d, will form the centerpiece of this review. In addition, we will DAscribe proteins that are putatively involved in kinetochore function specifically in the context with CBF3 interaction. Furthermore, we discuss the role of the S. cerevisiae kinetochores in a putative cell cycle checkpoint control and in microtubule attachment.

Biosynthesis of sulfated saccharides that are linked to asparagine residues in the cell surface glycoprotein of Halobacterium halobium via a glucose residue involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates (Lechner, J., Wieland, F., and Sumper, M. (1985) J. Biol. Chem. 260, 860-866). During isolation and characterization of these lipid oligosaccharides we detected a group of related compounds containing additional unidentified sugar residues. Here we report that: 1) the unknown sugar residues were 3-O-methylglucose, linked peripherally to the lipid-saccharide intermediates; 2) the 3-O-methylglucose residues in the oligosaccharides occur only at the lipid-linked level but are absent at the protein-linked level; 3) cell surface glycoprotein biosynthesis in Halobacteria in vivo is drastically depressed when S-adenosylmethionine-dependent methylation is inhibited, indicating that methylation is an obligatory step during glycoprotein synthesis. We propose a mechanism for the transport of lipid oligosaccharides through the cell membrane, involving an intermediate stage in which the saccharide moieties are transiently modified with 3-O-methylglucose.

A novel type of N-glycosidic linkage, asparaginyl glucose, occurs in the cell surface glycoprotein of halobacteria (Wieland, F., Heitzer, R., and Schaefer, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5470-5474). Sulfated oligosaccharides containing glucuronic acids are attached to the polypeptide chain via this linkage. Here we describe the isolation and chemical characterization of lipid-linked precursors of these saccharides, and these have the following new features. Rather than the bacterial undecaprenol, a C60-dolichol is the carrier lipid. The oligosaccharide is bound to this lipid via a monophosphate, rather than a pyrophosphate bridge. Sulfation of the saccharides is completed while they are linked to lipid and does not occur after transfer of the saccharides to protein.