A5058515611- Fungal and yeast genetics research
- Cellular transport and secretion
- Ubiquitin and proteasome pathways
- Endoplasmic Reticulum Stress and Disease
- Monoclonal and Polyclonal Antibodies Research
- Biotin and Related Studies
- Plant Reproductive Biology
- Microtubule and mitosis dynamics
- Biofuel production and bioconversion
- Protein Kinase Regulation and GTPase Signaling
- Cellular Mechanics and Interactions
- Fermentation and Sensory Analysis
- Click Chemistry and Applications
- Genetic Neurodegenerative Diseases
- S100 Proteins and Annexins
- Advanced Proteomics Techniques and Applications
- Microbial Metabolic Engineering and Bioproduction
- RNA Research and Splicing
- Bioinformatics and Genomic Networks
- Chemical Synthesis and Analysis
- Cardiomyopathy and Myosin Studies
- Protein Structure and Dynamics
- Cancer-related Molecular Pathways
- Glycosylation and Glycoproteins Research
- Advanced Fluorescence Microscopy Techniques
Universität Ulm
2016-2025
Institute of Molecular Genetics
2013-2024
Max Planck Institute of Molecular Cell Biology and Genetics
2022
University of Münster
2007
FZI Research Center for Information Technology
2002-2005
École Polytechnique Fédérale de Lausanne
2003-2005
Karlsruhe Institute of Technology
2003-2004
Max Delbrück Center
1998-2002
Institut für biologische Forschung
2002
Max Planck Institute for Plant Breeding Research
2000
We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When C-terminal fragment ubiquitin (C(ub)) is expressed as fusion to reporter only if N-terminal (Nub) also same cell. This reconstitution native from its fragments, detectable cleavage assay, not observed with mutationally altered Nub. However, C(ub) and Nub...
A detection system for interactions between membrane proteins in vivo is described. The based on split-ubiquitin [Johnsson, N. & Varshavsky, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10340-10344]. Interaction two detected by proteolytic cleavage of a protein fusion. releases transcription factor, which activates reporter genes the nucleus. As result, interaction can be analyzed means colorimetric assay. We use endoplasmic reticulum as model system. Wbp1p and Ost1p are both subunits...
The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for exclusive extracellular on surfaces live cells large variety including fluorophores, ligands, or quantum dots. approach is based acyl carrier through post-translational modification catalyzed by phosphopantetheine transferase. specificity versatility should allow it become an important tool studying...
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of proteome. By applying two distinct convolutional neural networks images millions live yeast cells, we resolved proteome-level dynamics both concentration during cell cycle, with resolution ∼20 classes. We show that a quarter proteome displays periodicity, proteins tending to be controlled either at level or concentration, but not both. Distinct levels protein regulation are preferentially...
A general method for the covalent immobilization of fusion proteins is presented. The approach based on unusual mechanism human O6-alkylguanine-DNA alkyltransferase, which irreversibly transfers alkyl group from its substrate, alkylated or benzylated guanine, to a reactive cysteine residue. By attaching benzyl surface, hAGT immobilize themselves in specific and manner. specificity reaction with substrate even allows directly out cell extracts, making an attractive alternative currently used...
The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo. Cub, C-terminal half Ub, attached Sec63p, and Nub, N-terminal selection differently localized proteins yeast Saccharomyces cerevisiae. efficiency Nub Cub reassembly quasi-native Ub reflects proximity between Sec63-Cub Nub-labeled proteins. By using modified Ura3p as reporter that is released from local concentration Sec63-Cub-RUra3p different Nub-constructs could be translated...
Biochemical and partial sequence data reveal the two-domain structure of p36. A loose some 30 residues at amino-terminus contains phosphorylatable tyrosine binding site for p11 regulatory chain. The following p33 domain retains lipid-binding as well Ca2+ which influences spectral properties single tryptophan one tyrosine. combined covering about 25% molecule identify p36 a unique polypeptide.
The split-ubiquitin assay detects protein interactions in vivo . To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the into a generally applicable screen for binding partners of specific A library genomic Saccharomyces cerevisiae DNA fragments fused to N-terminal half ubiquitin was constructed transformed yeast strains carrying either or Tup1p as bait. Both were C-terminally extended by C-terminal followed modified Ura3p an arginine position...
We describe a method that can be used to produce equimolar amounts of two or more specific proteins in cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, reference protein and interest are synthesized as polyprotein separated by ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally nearly so, ubiquitin-specific processing proteases after last residue ubiquitin, producing bearing C-terminal applications such pulse-chase analysis, UPR technique...
Abstract The septins of the yeast Saccharomyces cerevisiae assemble into hetero-octameric rods by alternating interactions between neighboring G-domains or N- and C-termini, respectively. These polymerize end to apolar filaments, forming a ring beneath prospective new bud that expands during cell cycle an hourglass structure. finally splits cytokinesis double ring. Understanding these transitions as well plasticity higher order assemblies requires detailed knowledge underlying structures....
Abstract The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras Rho, do not mediate signals effectors through GTP and hydrolysis. Biochemical approaches addressing how why...
The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras Rho, do not mediate signals effectors through GTP and hydrolysis. Biochemical approaches addressing how why utilize...
We report on a method for the multicolor imaging of cell surface proteins which is based labeling carrier protein (CP) fusion with different fluorophores. In one application, generations can be sequentially labeled to discriminate between old and newly made copies. another fusions CPs selectively fluorophores in sample. Both applications open up new ways studying properties living cells.
The mechanisms of the coordinated assembly and disassembly septin/myosin ring is central for understanding polar growth cytokinesis in yeast other organisms. septin- myosin-binding protein Bni5p provides a dual function during formation rings. Early cell cycle, captures Myo1p at incipient bud site actively transforms it into higher-order structures. Additionally, stabilizes released from septins shortly before onset cytokinesis. If this dissociation artificially prevented, impaired untimely...
The split-ubiquitin technique was used to detect transient protein interactions in living cells. N ub , the N-terminal half of ubiquitin (Ub), fused Sec62p, a component translocation machinery endoplasmic reticulum ofSaccharomyces cerevisiae. C C-terminal Ub, terminus signal sequence. reconstitution quasi-native Ub structure from two halves and resulting cleavage by Ub-specific proteases at serve as gauge proximity between test proteins linked . Using this assay, we show that Sec62p is...
The protein import machinery of the peroxisome consists many proteins, collectively called peroxins. By applying split-ubiquitin technique we systematically tested pair-wise interactions between Nub- and Cub-labeled peroxins for first time in living cells yeast Saccharomyces cerevisiae. We found that Pex10p plays a central role interaction network by connecting ubiquitin conjugation enzyme Pex4p to other members machinery. A strain harboring deletion PEX3 enabled us estimate influence...
Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit channel that guides proteins across membrane endoplasmic reticulum. The split-ubiquitin technique was used determine whether serves as an additional vivo. We measured interactions between N ub -labeled C -translocation substrates bearing four different sequences. so-determined interaction profile compared with correspondingly modified -Sec61p. assay reveals Kar2p...