Advanced photoelectric effect experiment beamline at Elettra: A surface science laboratory coupled with Synchrotron Radiation.

We report the main characteristics of the advanced photoelectric effect experiments beamline, operational at Elettra storage ring, featuring a fully independent double branch scheme obtained by the use of chicane undulators and able to keep polarization control in both linear and circular mode. The paper describes the novel technical solutions adopted, namely, (a) the design of a quasiperiodic undulator resulting in optimized suppression of higher harmonics over a large photon energy range (10-100 eV), (b) the thermal stability of optics under high heat load via cryocoolers, and (c) the end station interconnected setup allowing full access to off-beam and on-beam facilities and, at the same time, the integration of users' specialized sample growth chambers or modules.

Evidence for a magnetic proximity effect up to room temperature at Fe/(Ga, Mn)As interfaces.

We report x-ray magnetic circular dichroism and superconducting quantum interference device magnetometry experiments to study magnetic order and coupling in thin Fe/(Ga, Mn)As(100) films. We observe induced magnetic order in the (Ga, Mn)As layer that extends over more than 2 nm, even at room temperature. We find spectroscopic evidences of a hybridized d configuration of Mn atoms in Fe/(Ga, Mn)As, with negligible Mn diffusion and/or MnFe intermixing. We show by experiment as well as by theory that the magnetic moment of the Mn ions couples antiparallel to the moment of the Fe overlayer.

Molecular genetics of the ubiquitin-proteasome system: lessons from yeast.

Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATalpha2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to alpha2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as alpha2. The 26S proteasome is responsible for alpha2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.

Three-dimensional tomography of the beryllium fermi surface: surface charge redistribution.

The discontinuity in the lattice periodic potential at surfaces often leads to the creation of new electronic surface states. We developed a photoemission based Fermi surface tomography whose surface sensitivity allowed us to quantify the charge redistribution on the Be(0001) surface. The volume enclosed by the bulklike Fermi surface is significantly reduced at the surface, consistent with the charge transfer to the two surface states as estimated from the area within their two-dimensional Fermi contours. This result represents the first quantification of the charge redistribution on a natural surface termination.

Spin-resolved photoemission of surface states of W(110)-(1 x 1)H.

The surface electronic states of W(110)-(1 x 1)H have been measured using spin- and angle-resolved photoemission. We directly demonstrate that the surface bands are both split and spin-polarized by the spin-orbit interaction in association with the loss of inversion symmetry near a surface. We observe 100% spin polarization of the surface states, with the spins aligned in the plane of the surface and oriented in a circular fashion relative to the Smacr; symmetry point. In contrast, no measurable polarization of nearby bulk states is observed.

SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.

SUMO is covalently linked to a variety of cellular proteins. Three groups now describe related E3-like factors that enhance transfer of SUMO to specific proteins. This family of factors includes proteins important for chromosome condensation, signal transduction, and ion channel biogenesis.

A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.

Substrate discrimination in the ubiquitin-proteasome system is believed to be dictated by specific combinations of ubiquitin-protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Here we identify Doa10/Ssm4 as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Matalpha2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of alpha2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1-bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10Delta hrd1Delta mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder.

Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae.

Attachment of proteins to ubiquitin is reversed by specialized proteases called deubiquitinating enzymes (Dubs), which are also essential for ubiquitin precursor processing. In the genome of Saccharomyces cerevisiae, 17 potential DUB genes can be discerned. We have now constructed strains deleted for each of these genes. Surprisingly, given the essential nature of the ubiquitin system, none of the mutants is lethal or strongly growth defective under standard conditions, although a number have detectable abnormalities. Including results from this study, 14 of the 17 Dubs have now been shown to have ubiquitin-cleaving activity. The most extensively characterized yeast Dub is Doa4, which is required for both ubiquitin homeostasis and proteasome-dependent proteolysis. To help determine what distinguishes Doa4 functionally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyveromyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage. The data suggest that the DOA4 locus underwent a duplication after the divergence of K. lactis and S. cerevisiae. This information will facilitate fine-structure analysis of the Doa4 protein to help delineate its key functional elements.

The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways.

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

Biochemistry. All in the ubiquitin family.

The job of a protein can be altered by addition of molecules such as ubiquitin or the related ubiquitin-like modifiers, which bring about changes in the protein's localization, conformation, or its interactions with other proteins. In a comprehensive Perspective, Hochstrasser brings us up to date with the many new members of the ubiquitin modifier family and their multitudinous and diverse protein targets.

Evolution and function of ubiquitin-like protein-conjugation systems.

Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system.

A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in Saccharomyces cerevisiae.

Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin-proteasome pathway substrates as well as ubiquitin-dependent but proteasome-independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin-dependence of cellular processes in yeast, even those in which the proteasome is not involved.

The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein.

Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. Like SMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1(ts) strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Delta mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of a ulp1(ts) mutation or overproduction of catalytically inactive Ulp1 can substantially overcome the ulp2Delta defects. Inactivation of Ulp2 also suppresses several ulp1(ts) defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

The Saccharomyces cerevisiae ubiquitin-proteasome system.

Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MAT alpha 2. The alpha 2 protein is polyubiquitinated and rapidly degraded in alpha-haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum-localized ubiquitin-conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in alpha 2. Interestingly, degradation of alpha 2 is blocked in a/alpha-diploid cells by heterodimer formation between the alpha 2 and a1 homeodomain proteins. The data suggest that degradation signals may overlap protein-protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of alpha 2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active-site formation were subsequently uncovered. Finally, it has become clear that protein (poly) ubiquitination is highly dynamic in vivo, and our studies of yeast de-ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast.

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.

Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome. The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings. Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1. Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly. We show here that the propeptides have both common and distinct functions in proteasome biogenesis. Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation. However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly. Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation. For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated. In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism.

Structure and functional analysis of the 26S proteasome subunits from plants.

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN 10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10delta strains which grow normally, Physcomitrella rpn10delta strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.

A new protease required for cell-cycle progression in yeast.

In eukaryotes, protein function can be modulated by ligation to ubiquitin or to ubiquitin-like proteins (Ubl proteins). The vertebrate Ubl protein SUMO-1 is only 18% identical to ubiquitin but is 48% identical to the yeast protein Smt3. Both SUMO-1 and Smt3 are ligated to cellular proteins, and protein conjugation to SUMO-1/Smt3 is involved in many physiological processes. It remained unknown, however, whether deconjugation of SUMO-1/Smt3 from proteins is also essential. Here we describe a yeast Ubl-specific protease, Ulp1, which cleaves proteins from Smt3 and SUMO-1 but not from ubiquitin. Ulp1 is unrelated to any known deubiquitinating enzyme but shows distant similarity to certain viral proteases, indicating the existence of a widely conserved protease fold. Proteins related to Ulp1 are present in many organisms, including several human pathogens. The pattern of Smt3-coupled proteins in yeast changes markedly throughout the cell cycle, and specific conjugates accumulate in ulp1 mutants. Ulp1 has several functions, including an essential role in the G2/M phase of the cell cycle.