Julie Huang

An Enzymatic Activity in the Yeast Sir2 Protein that Is Essential for Gene Silencing

Despite its conservation in organisms from bacteria to human and its general requirement for transcriptional silencing in yeast, the function of the Sir2 protein is unknown. Here we show that Sir2 can transfer labeled phosphate from nicotinamide adenine dinucleotide to itself and histones in vitro. A modified form of Sir2, which results from its automodification activity, is specifically recognized by anti-mono-ADP-ribose antibodies, suggesting that Sir2 is an ADP-ribosyltransferase. Mutation of a phylogenetically invariant histidine residue in Sir2 abolishes both its enzymatic activity in vitro and its silencing functions in vivo. However, the mutant protein is associated with chromatin and other silencing factors in a manner similar to wild-type Sir2. These findings suggest that Sir2 contains an ADP-ribosyltransferase activity that is essential for its silencing function.

Lis1 Acts as a ''Clutch'' between the ATPase and Microtubule-Binding Domains of the Dynein Motor

Summary The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dyneins AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a “clutch” that prevents dyneins ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments.

Structural Basis for Microtubule Binding and Release by Dynein

Motoring Along Dyneins are large and complex molecular motors that transport cargo along cellular microtubules and power the movement of cilia. An enigma is how microtubule binding and nucleotide hydrolysis are coordinated between sites separated by 25 nm. Redwine et al. (p. 1532) report an electron microscopy structure of the dynein microtubule-binding domain bound to microtubules in a high-affinity state and combined this with molecular dynamics and existing x-ray structures to provide a model for how dynein couples its affinity for microtubules with the nucleotide-bound state of the motor domain. The molecular motor dynein uses conformational changes within its microtubule-binding domain to modulate track affinity. Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein’s track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein’s microtubule-binding domain bound to microtubules by cryo–electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein’s affinity for microtubules. Our results provide a molecular model for how dynein’s binding to microtubules is communicated to the rest of the motor.

The Replication Fork Block Protein Fob1 Functions as a Negative Regulator of the FEAR Network

BACKGROUND The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase. RESULTS Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1. CONCLUSIONS Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.

Evaluation of the Nucleolar Localization of the RENT Complex to Ribosomal DNA by Chromatin Immunoprecipitation Assays

Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein-DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i.e., ribosomal DNA (rDNA).