Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background: The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported. Methodology/Principal Findings: In the present study we found that asc1-deletion mutant (asc1D) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1D cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells ’ survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1D/pkc1D) to cell-wall stress conditions, and high basal level of PKC signaling in asc1D. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions. Conclusions/Significance: Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p act

Physical structure of nuclear receptor-DNA complexes

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Structural basis of RXR-DNA interactions

The 9-cis retinoic acid receptor, RXR, binds DNA effectively as a homodimer or as a heterodimer with other nuclear receptors. The DNA-binding sites for these RXR complexes are direct repeats of a consensus sequence separated by one to five base-pairs of intervening space. Here, we report the 2.1 A crystal structure of the RXR-DNA-binding domain as a homodimer in complex with its idealized direct repeat DNA target. The structure shows how a gene-regulatory site can induce conformational changes in a transcription factor that promote homo-cooperative assembly. Specifically, an alpha-helix in the T-box is disrupted to allow efficient DNA-binding and subunit dimerization. RXR displays a relaxed mode of sequence recognition, interacting with only three base-pairs in each hexameric half-site. The structure illustrates how site selection is achieved in this large eukaryotic transcription factor family through discrete protein-protein interactions and the use of tandem DNA binding sites with characteristic spacings.status: publishe

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment