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Günter Blobel

Publications and source records attributed to Günter Blobel.

13 recordsLinked to original sources
PubMed2006

Rae1 interaction with NuMA is required for bipolar spindle formation.

In eukaryotic cells, the faithful segregation of daughter chromosomes during cell division depends on formation of a microtubule (MT)-based bipolar spindle apparatus. The Nuclear Mitotic Apparatus protein (NuMA) is recruited from interphase nuclei to spindle MTs during mitosis. The carboxy terminal domain of NuMA binds MTs, allowing a NuMA dimer to function as a "divalent" crosslinker that bundles MTs. The messenger RNA export factor, Rae1, also binds to MTs. Lowering Rae1 or increasing NuMA levels in cells results in spindle abnormalities. We have identified a mitotic-specific interaction between Rae1 and NuMA and have explored the relationship between Rae1 and NuMA in spindle formation. We have mapped a specific binding site for Rae1 on NuMA that would convert a NuMA dimer to a "tetravalent" crosslinker of MTs. In mitosis, reducing Rae1 or increasing NuMA concentration would be expected to alter the valency of NuMA toward MTs; the "density" of NuMA-MT crosslinks in these conditions would be diminished, even though a threshold number of crosslinks sufficient to stabilize aberrant multipolar spindles may form. Consistent with this interpretation, we found that coupling NuMA overexpression to Rae1 overexpression or coupling Rae1 depletion to NuMA depletion prevented the formation of aberrant spindles. Likewise, we found that overexpression of the specific Rae1-binding domain of NuMA in HeLa cells led to aberrant spindle formation. These data point to the Rae1-NuMA interaction as a critical element for normal spindle formation in mitosis.

Antigens, Nuclear↗
PubMed2006

Karyopherin-mediated import of integral inner nuclear membrane proteins.

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.

Amino Acid Motifs↗
PubMed2006

Crystal structure and mechanism of human lysine-specific demethylase-1.

The reversible methylation of specific lysine residues in histone tails is crucial in epigenetic gene regulation. LSD1, the first known lysine-specific demethylase, selectively removes monomethyl and dimethyl, but not trimethyl modifications of Lys4 or Lys9 of histone-3. Here, we present the crystal structure of LSD1 at 2.9-A resolution. LSD1 forms a highly asymmetric, closely packed domain structure from which a long helical 'tower' domain protrudes. The active site cavity is spacious enough to accommodate several residues of the histone tail substrate, but does not appear capable of recognizing the different methylation states of the substrate lysine. This supports the hypothesis that trimethylated lysine is chemically rather than sterically discriminated. We present a biochemical analysis of LSD1 mutants that identifies crucial residues in the active site cavity and shows the importance of the SWIRM and tower domains for catalysis.

Binding Sites↗
PubMed2006

Homodimerization of the G protein SRbeta in the nucleotide-free state involves proline cis/trans isomerization in the switch II region.

Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the alpha- and beta-subunits of SR. We have determined the 2.2-A crystal structure of the nucleotide-free SRbeta domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SRalpha binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.

Amino Acid Sequence↗
PubMed2005

A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization.

Through a genetic screen using myosin-like protein strains mlp1Delta mlp2Delta and biochemical purification, we identified a complex of eight proteins, each required for growth and DNA repair in Saccharomyces cerevisiae. Among the subunits are Mms21 that contains a putative Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain characteristic of small ubiquitin-like modifier (SUMO) ligases, two structural-maintenance-of-chromosome (Smc) proteins, Smc5 and Smc6, and a protein that contains an ubiquitin ligase signature domain. We show that these proteins colocalized to several distinct nuclear foci. Biochemical and genetic data demonstrated that Mms21 indeed functions as a SUMO ligase and that this activity requires the Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain. The substrates for this SUMO ligase include a subunit of the octameric complex, Smc5, and the DNA repair protein Yku70. We further show that the abolition of the SUMO E3 activity of Mms21 leads to such disparate phenotypes as DNA damage sensitivity, defects in nucleolar integrity and telomere clustering, silencing, and length regulation. We propose that Mms21 sumoylates proteins involved in these diverse processes and that the other members of the complex, particularly Smc5/6, facilitate proper substrate sumoylation by localizing Mms21 to specific chromosomal regions.

Amino Acid Sequence↗
PubMed2005

Carrier-independent nuclear import of the transcription factor PU.1 via RanGTP-stimulated binding to Nup153.

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

Active Transport, Cell Nucleus↗
PubMed2004

Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality.

Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

Clone Cells↗
PubMed2004

Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

Active Transport, Cell Nucleus↗
PubMed2004

Circular permutation as a tool to reduce surface entropy triggers crystallization of the signal recognition particle receptor beta subunit.

The production of diffraction-quality crystals remains a difficult obstacle on the road to high-resolution structural characterization of proteins. This is primarily a result of the empirical nature of the process. Although crystallization is not predictable, factors inhibiting it are well established. First, crystal formation is always entropically unfavorable. Reducing the entropic cost of crystallizing a given protein is thus desirable. It is common practice to map boundaries and remove unstructured regions surrounding the folded protein domain. However, a problem arises when flexible regions are not at the boundaries but within a domain. Such regions cannot be deleted without adding new restraints to the domain. We encountered this problem during an attempt to crystallize the beta subunit of the eukaryotic signal recognition particle (SRbeta), bearing a long and flexible internal loop. Native SRbeta did not crystallize. However, after circularly permuting the protein by connecting the spatially close N and C termini with a short heptapeptide linker GGGSGGG and removing 26 highly flexible loop residues within the domain, we obtained diffraction-quality crystals. This protein-engineering method is simple and should be applicable to other proteins, especially because N and C termini of protein domains are often close in space. The success of this method profits from prior knowledge of the domain fold, which is becoming increasingly common in today's postgenomic era.

Crystallization↗
PubMed2003

Structural basis for the function of the beta subunit of the eukaryotic signal recognition particle receptor.

Protein translocation across and insertion into membranes is a process essential to all life forms. In higher eukaryotes, this process is initiated by targeting the translating ribosome to the endoplasmic reticulum via the signal recognition particle (SRP) and its membrane-associated heterodimeric receptor (SR). This targeting step is regulated by three G proteins, SRP54, SR alpha, and SR beta, which act in concert. Little is known about the regulatory role of SR beta. Here, we present the 1.7 A crystal structure of the SR beta-GTP subunit in complex with the interaction domain of SR alpha. Strikingly, the binding interface overlaps largely with the switch 1 region of SR beta. This finding, together with additional biochemical data, shows that the eukaryotic SR is a conditional and not an obligate heterodimer. The results suggest that the GTP/GDP switch cycle of SR beta functions as a regulatory switch for the receptor dimerization. We discuss the implications for the translocation pathway.

Amino Acid Sequence↗
PubMed2002

Uncoupling Kapbeta2 substrate dissociation and ran binding.

Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.

Active Transport, Cell Nucleus↗
PubMed2002

Role of nucleoporin induction in releasing an mRNA nuclear export block.

Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC). Some NPC components, such as the nucleoporins (Nups) Nup98 and Nup96, are also associated with the nuclear interior. Nup98 is a target of the vesicular stomatitis virus (VSV) matrix (M) protein-mediated inhibition of messenger RNA (mRNA) nuclear export. Here, Nup98 and Nup96 were found to be up-regulated by interferon (IFN). M protein-mediated inhibition of mRNA nuclear export was reversed when cells were treated with IFN-gamma or transfected with a complementary DNA (cDNA) encoding Nup98 and Nup96. Thus, increased Nup98 and Nup96 expression constitutes an IFN-mediated mechanism that reverses M protein-mediated inhibition of gene expression.

Active Transport, Cell Nucleus↗