The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae. The split-ubiquitin method is based on the ability of N ub and C ub , the N-and C-terminal halves of ubiquitin, to form a native-like ubiquitin (1). Ubiquitin-specific proteases (UBPs), present in the cytosol and nucleus of all eukaryotic cells, recognize the reconstituted ubiquitin, but not its halves, and cleave off a reporter protein, which had been linked to the C terminus of C ub . The split-ubiquitin assay (split-Ub) is designed to yield efficient association of N ub and C ub only if the two ubiquitin halves are linked to proteins that interact in vivo. The assay has been shown to detect interactions between cytosolic proteins, membrane proteins, and transient interactions that occur between transporter and substrate during protein translocation across the membrane of the endoplasmic reticulum in vivo (1-4). In addition, split-Ub can also be used to demonstrate interactions between transcription factors (5, 6) because, contrary to the two-hybrid system (7), it is not based on a transcriptional readout.The Saccharomyces cerevisiae GAL1 promoter is a wellstudied example of transcriptional regulation by nutrients. When the cells are grown in medium containing galactose as the sole carbon source, GAL1 is activated by Gal4p, which binds specifically to the GAL1 promoter. Gal4p interacts with the holoenzyme component Srb4p, thereby recruiting the transcription apparatus to the GAL1 promoter (8). If the carbon source is switched to glucose, the promoter is repressed by two independently operating mechanisms. Gal80p masks the activation domain of DNA-bound Gal4p, thereby preventing the recruitment of the transcription machinery (9). In addition, the cytosolic repressor Mig1p enters the nucleus (10). Mig1p b...

Slow Wallerian degeneration (Wld S ) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD؉ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld S targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld S lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD ؉ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld S protein influence the intranuclear location of both ubiquitin proteasome and NAD ؉ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP. INTRODUCTIONThe E4 ubiquitination factor Ube4b (or Ufd2a) has a 123-amino acid N-terminal region that is essential for ubiquitination activity (Mahoney et al., 2002). It is unclear why this region is essential because it does not contain the U box, and it appears to be absent in invertebrate orthologues that ubiquitinate effectively (Koegl et al., 1999;Hatakeyama et al., 2001;Mahoney et al., 2002;Hoppe et al., 2004;Richly et al., 2005). It is important to understand the molecular mechanism of Ube4b because it has a key role in the ubiquitin proteasome system (UPS; Hoppe, 2005), it is neuroprotective in polyglutamine disorders and an important candidate gene for neuroblastoma (Krona et al., 2003). Information on the substrates of Ube4b is beginning to emerge (Hoppe et al., 2004;Okumura et al., 2004;Spinette et al., 2004;Richly et al., 2005) but there is much still to learn about its regulation.In the slow Wallerian degeneration mutant mouse (Wld S ), 70 amino acids of this essential domain of Ube4b form the N-terminus of a chimeric protein that delays Wallerian degeneration of injured axons in mice and rats by 10-fold (see Figure 1A; Lunn et al., 1989;Mack et al., 2001;Adalbert et al., 2005). The chimeric protein is absent in wild-type mice. This sequence (N70) is fused in Wld S protein to the full coding sequence of nicotinamide mononucleotide adenylyltransferase (Nmnat1; Conforti et al., 2000;Emanuelli et al., 2001;Mack et al., 2001) in regulating axon degeneration. Wld S also delays axon degeneration in a wide range of neurodegenerative disorders and acute retrograde axonal degeneration after spinal injury, indicating that axon degeneration mechanisms are more closely related th...

Proteasome inhibitors such as lactacystin were first isolated when assaying their ability to stimulate neurite outgrowth in neuronal-like cell lines; however, their effect on neurites in primary culture has been largely neglected. We report here that lactacystin causes immediate arrest of nerve growth factor (NGF)-stimulated neurite outgrowth in sympathetic and sensory explant cultures. This is followed by neurite degeneration that in sympathetic cultures has a distinctive "dying-back" morphology. Remarkably, this occurs even at concentrations below that required to induce neurite outgrowth in PC12 cells. Thus, lactacystin opposes rather than potentiates the effect of NGF on sympathetic neurite outgrowth and the role of the ubiquitin proteasome pathway in growth and long-term maintenance of axons and dendrites differs from that in neuritogenesis in neuronal-like cell lines. Retrograde degeneration caused by blocking of the ubiquitin proteasome pathway may mimic some aspects of gracile axonal dystrophy, a dying-back axonopathy in mice caused by ubiquitin hydrolase (Uch-l1) deficiency, and may be relevant to human neurodegenerative diseases involving ubiquitination or proteasome abnormalities.

AcKnowLedGementsThis work has been supported by DFG JE 252/4 and the BMBF BioFuture program. Thanks are due to A. Pingoud for helpful discussions and M. Schwerdtfeger for technical assistance. AbstRActDNA methyltransferase 1 methylates hemi-methylated CG sites generated during DNA replication. Serine 515 of this enzyme has been shown to be phosphorylated. To explore the importance of S515 phosphorylation, we generated mutants of Dnmt1 which removed the phosphorylation potential (S515A) or mimic phosphoserine (S515E), purified the proteins from insect cells and analyzed their DNA methylation activity in vitro. The S515E mutant was found to be active, while S515A mutant had severe loss in activity when compared to the wild type protein. The loss of activity of the S515A variant was not due to loss of DNA binding capacity. Furthermore, we show that a phosphorylated peptide whose sequence mimics the surrounding of Ser515 (EKIYIS P KIVVE) inhibited the activity of wild type Dnmt1 ten-fold more than the non-phosphorylated peptide. The inhibition was specific for Dnmt1 and for the particular peptide sequence. Our data suggest that phosphorylation of Ser515 is important for an interaction between the N-terminal domain of Dnmt1 and its catalytic domain that is necessary for activity and that this interaction is specifically disrupted by the phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515 could be an important regulator of Dnmt1 activity during cell cycle and after proliferative stimuli.

Protein-protein interactions are critical to most biological processes, and locating protein-protein interfaces on protein structures is an important task in molecular biology. We developed a new experimental strategy called the 'absence of interference' approach to determine surface residues involved in protein-protein interaction of established yeast two-hybrid pairs of interacting proteins. One of the proteins is subjected to high-level randomization by error-prone PCR. The resulting library is selected by yeast two-hybrid system for interacting clones that are isolated and sequenced. The interaction region can be identified by an absence or depletion of mutations. For data analysis and presentation, we developed a Web interface that analyzes the mutational spectrum and displays the mutational frequency on the surface of the structure (or a structural model) of the randomized protein †. Additionally, this interface might be of use for the display of mutational distributions determined by other types of random mutagenesis experiments. We applied the approach to map the interface of the catalytic domain of the DNA methyltransferase Dnmt3a with its regulatory factor Dnmt3L. Dnmt3a was randomized with high mutational load. A total of 76 interacting clones were isolated and sequenced, and 648 mutations were identified. The mutational pattern allowed to identify a unique interaction region on the surface of Dnmt3a, which comprises about 500−600 Å 2 . The results were confirmed by site-directed mutagenesis and structural analysis. The absence-of-interference approach will allow high-throughput mapping of protein interaction sites suitable for functional studies and protein docking.