Latest Articles Include:
- Amino Acid Signaling to TOR Activation: Vam6 Functioning as a Gtr1 GEF
- Mol Cell 35(5):543-545 (2009)
In this issue of Molecular Cell, Binda et al. identify Vam6/Vps39 as a guanine nucleotide exchange factor for Gtr1, a Rag family GTPase, to promote TORC1 activation in response to amino acids.
- The Alternating Power Stroke of a 6-Cylinder AAA Protease Chaperone Engine
- Mol Cell 35(5):545-547 (2009)
In this issue ofMolecular Cell, Augustin et al. (2009) describe the regulatory coupling of adjacent ATP subunits in the mitochondrial AAA protease ring along with the responsible molecular determinants and uncover its importance for membrane dislocation of substrates.
- Resolving the CSN and CAND1 Paradoxes
- Mol Cell 35(5):547-549 (2009)
In this issue of Molecular Cell, Schmidt et al. (2009) untangle the interplay between the COP9 signalosome (CSN), cullin-associated and neddylation-dissociated 1 (CAND1) protein, and cullin-RING ubiquitin ligases (CRLs).
- XPD Helicase Speeds through a Molecular Traffic Jam
- Mol Cell 35(5):549-550 (2009)
Helicases and other DNA translocases must travel along crowded substrates. In this issue, Honda et al. (2009) report that the archaeal XPD helicase can bypass a single-stranded DNA-binding protein without either molecule being ejected from the DNA.
- Fine-Tuning of the Unfolded Protein Response: Assembling the IRE1α Interactome
- Mol Cell 35(5):551-561 (2009)
Endoplasmic reticulum (ER) stress is a hallmark feature of secretory cells and many diseases, including cancer, neurodegeneration, and diabetes. Adaptation to protein-folding stress is mediated by the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). The UPR signals through three distinct stress sensors located at the ER membrane-IRE1α, ATF6, and PERK. Although PERK and IRE1α share functionally similar ER-luminal sensing domains and both are simultaneously activated in cellular paradigms of ER stress in vitro, they are selectively engaged in vivo by the physiological stress of unfolded proteins. The differences in terms of tissue-specific regulation of the UPR may be explained by the formation of distinct regulatory protein complexes. This concept is supported by the recent identification of adaptor and modulator proteins that directly interact with IRE1α. In this Review, we discuss recent evidence supporting a mod! el where IRE1α signaling emerges as a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble.
- The Vam6 GEF Controls TORC1 by Activating the EGO Complex
- Mol Cell 35(5):563-573 (2009)
The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1GTP, which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1GDP, caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating th! e Gtr1 subunit of the EGO complex.
- An Intersubunit Signaling Network Coordinates ATP Hydrolysis by m-AAA Proteases
- Mol Cell 35(5):574-585 (2009)
Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit, leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates, but not for substrate processing. These findings provide insight into how AAA+ proteins convert energy derived from ATP hydrolysis into mechani! cal work.
- F-Box-Directed CRL Complex Assembly and Regulation by the CSN and CAND1
- Mol Cell 35(5):586-597 (2009)
The COP9 signalosome (CSN) is thought to maintain the stability of cullin-RING ubiquitin ligases (CRL) by limiting the autocatalytic destruction of substrate adapters such as F box proteins (FBPs). CAND1, a protein associated with unneddylated CUL1, was proposed to assist in this role in an as yet unclear fashion. We found that only a subset of Schizosaccharomyces pombe FBPs, which feature a critical F box proline that promotes their interaction with CUL1, required CSN for stability. Unlike the CRL3 adaptor Btb3p, none of the CSN-sensitive FBPs were affected by deletion of ubp12. Contrary to current models, CAND1 does not control adaptor stability but maintains the cellular balance of CRL1 complexes by preventing rare FBPs from being outcompeted for binding to CUL1 by more ample adapters. These findings were integrated into a refined model of CRL control in which substrate availability toggles CRLs between independent CSN and CAND1 cycles.
- Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA
- Mol Cell 35(5):598-609 (2009)
The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF3−, or ADP-AlF4−. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge α helix bending RNA 3′ of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5′ of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which! a protein uses two wedges to act as a molecular crimper.
- miR-24 Inhibits Cell Proliferation by Targeting E2F2, MYC, and Other Cell-Cycle Genes via Binding to "Seedless" 3′UTR MicroRNA Recognition Elements
- Mol Cell 35(5):610-625 (2009)
miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance (MYC, E2F2, CCNB1, and CDC2) or inhibit (p27Kip1 and VHL) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2, but not MYC, and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2. Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2, MY! C, AURKB, CCNA2, CDC2, CDK4, and FEN1 by recognizing seedless but highly complementary sequences.
- Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation
- Mol Cell 35(5):626-641 (2009)
To identify regulators involved in determining the differential pattern of H3K79 methylation by Dot1, we screened the entire yeast gene deletion collection by GPS for genes required for normal levels of H3K79 di- but not trimethylation. We identified the cell cycle-regulated SBF protein complex required for H3K79 dimethylation. We also found that H3K79 di- and trimethylation are mutually exclusive, with M/G1 cell cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is not detected on cell cycle-regulated genes and sites containing H3K79me2, but is found on H3K79me3-containing regions. A screen for genes responsible for the establishment/removal of H3K79 dimethylation resulted in identification of NRM1 and WHI3, both of which impact the transcription by the SBF and MBF protein co! mplexes, further linking the regulation of methylation status of H3K79 to the cell cycle.
- The Interaction of NSBP1/HMGN5 with Nucleosomes in Euchromatin Counteracts Linker Histone-Mediated Chromatin Compaction and Modulates Transcription
- Mol Cell 35(5):642-656 (2009)
Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcrip! tion profile by counteracting the chromatin-condensing activity of linker histones.
- Structural and Functional Insights into the Roles of the Mms21 Subunit of the Smc5/6 Complex
- Mol Cell 35(5):657-668 (2009)
The Smc5/6 complex is an evolutionarily conserved chromosomal ATPase required for cell growth and DNA repair. Its Mms21 subunit supports both functions by docking to the arm region of Smc5 and providing SUMO ligase activity. Here, we report the crystal structure of Mms21 in complex with the Smc5 arm. Our structure revealed two distinct structural and functional domains of the Smc5-bound Mms21: its N-terminal half is dedicated to Smc5 binding by forming a helix bundle with a coiled-coil structure of Smc5; its C-terminal half includes the SUMO ligase domain, which adopts a new type of RING E3 structure. Mutagenesis and structural analyses showed that the Mms21-Smc5 interface is required for cell growth and resistance to DNA damage, while the unique Mms21 RING domain confers specificity to the SUMO E2-E3 interaction. Through structure-based dissection of Mms21 functions, our studies establish a framework for understanding its roles in the Smc5/6 complex.
- Structure of the Siz/PIAS SUMO E3 Ligase Siz1 and Determinants Required for SUMO Modification of PCNA
- Mol Cell 35(5):669-682 (2009)
Siz1 is a founding member of the Siz/PIAS RING family of SUMO E3 ligases. The X-ray structure of an active Siz1 ligase revealed an elongated tripartite architecture comprised of an N-terminal PINIT domain, a central zinc-containing RING-like SP-RING domain, and a C-terminal domain we term the SP-CTD. Structure-based mutational analysis and biochemical studies show that the SP-RING and SP-CTD are required for activation of the E2SUMO thioester, while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a nonconsensus lysine residue that is not modified by the SUMO E2 in the absence of Siz1. Mutational analysis of Siz1 and PCNA revealed surfaces on both proteins that are required for efficient SUMO modification of PCNA in vitro and in vivo.
- The Effect of Replication Initiation on Gene Amplification in the rDNA and Its Relationship to Aging
- Mol Cell 35(5):683-693 (2009)
In eukaryotes, the ribosomal DNA (rDNA) consists of long tandem repeat arrays. These repeated genes are unstable because homologous recombination between them results in copy number loss. To maintain high copy numbers, yeast has an amplification system that works through a pathway involving the replication fork barrier site and unequal sister chromatid recombination. In this study, we show that an active replication origin is essential for amplification, and the amplification rate correlates with origin activity. Moreover, origin activity affects the levels of extrachromosomal rDNA circles (ERC) that are thought to promote aging. Surprisingly, we found that reduction in ERC level results in shorter life span. We instead show that life span correlates with rDNA stability, which is preferentially reduced in mother cells, and that episomes can induce rDNA instability. These data support a model in which rDNA instability itself is a cause of aging in yeast.
- Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase
- Mol Cell 35(5):694-703 (2009)
An encounter between a DNA-translocating enzyme and a DNA-bound protein must occur frequently in the cell, but little is known about its outcome. Here we developed a multicolor single-molecule fluorescence approach to simultaneously monitor single-stranded DNA (ssDNA) translocation by a helicase and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA-binding properties, the two cognate ssDNA-binding proteins RPA1 and RPA2 differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.
- Checkpoint Signaling from a Single DNA Interstrand Crosslink
- Mol Cell 35(5):704-715 (2009)
DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivit! y to crosslinking agents results from both checkpoint and DNA repair defects.
- Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites Is Differentially Governed by Replication
- Mol Cell 35(5):716-723 (2009)
Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remain unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-rel! ated FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status.