Comprehensive proteomics analysis reveals new substrates and regulators of the fission yeast clp1/cdc14 phosphatase.

The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear ß-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms.

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

State of the APC/C: organization, function, and structure.

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response. The anaphase-promoting complex/cyclosome (APC/C), an evolutionarily conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis.

A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity.

Ubiquitination and deubiquitination are reciprocal processes that tune protein stability, function, and/or localization. The removal of ubiquitin and remodeling of ubiquitin chains is catalyzed by deubiquitinating enzymes (DUBs), which are cysteine proteases or metalloproteases. Although ubiquitination has been extensively studied for decades, the complexity of cellular roles for deubiquitinating enzymes has only recently been explored, and there are still several gaps in our understanding of when, where, and how these enzymes function to modulate the fate of polypeptides. To address these questions we performed a systematic analysis of the 20 Schizosaccharomyces pombe DUBs using confocal microscopy, proteomics, and enzymatic activity assays. Our results reveal that S. pombe DUBs are present in almost all cell compartments, and the majority are part of stable protein complexes essential for their function. Interestingly, DUB partners identified by our study include the homolog of a putative tumor suppressor gene not previously linked to the ubiquitin pathway, and two conserved tryptophan-aspartate (WD) repeat proteins that regulate Ubp9, a DUB that we show participates in endocytosis, actin dynamics, and cell polarity. In order to understand how DUB activity affects these processes we constructed multiple DUB mutants and find that a quintuple deletion of ubp4 ubp5 ubp9 ubp15 sst2/amsh displays severe growth, polarity, and endocytosis defects. This mutant allowed the identification of two common substrates for five cytoplasmic DUBs. Through these studies, a common regulatory theme emerged in which DUB localization and/or activity is modulated by interacting partners. Despite apparently distinct cytoplasmic localization patterns, several DUBs cooperate in regulating endocytosis and cell polarity. These studies provide a framework for dissecting DUB signaling pathways in S. pombe and may shed light on DUB functions in metazoans.

Survey of the phosphorylation status of the Schizosaccharomyces pombe deubiquitinating enzyme (DUB) family.

Ubiquitination plays a role in virtually every cellular signaling pathway ranging from cell cycle control to DNA damage response to endocytosis and gene regulation. The bulk of our knowledge of the ubiquitination system is centered on modification of specific substrate proteins and the enzymatic cascade of ubiquitination. Our understanding of the regulation of the reversal of these modifications (deubiquitination) lags significantly behind. We recently reported a multifaceted study of the fission yeast Schizosaccharomyces pombe DUBs including characterization of their binding partners, in vitro enzymatic activity and subcellular localization. (1) Over half of the 20 fission yeast DUBs have a stable protein partner and some of those partners regulate the localization and/or activity of their cognate DUB. As a next step in understanding how DUBs might otherwise be regulated, we investigated the phosphostatus of the entire fission yeast DUB family using LC-MS/MS, and here we discuss the possible implications of phosphoregulation.

A collision cross-section database of singly-charged peptide ions.

A database of ion-neutral collision cross-sections for singly-charged peptide ions is presented. The peptides included in the database were generated by enzymatic digestion of known proteins using three different enzymes, resulting in peptides that differ in terms of amino acid composition as well as N-terminal and C-terminal residues. The ion-neutral collision cross-sections were measured using ion mobility (IM) spectrometry that is directly coupled to a time-of-flight (TOF) mass spectrometer. The ions were formed by a matrix-assisted laser desorption ionization (MALDI) ion source operated at pressures (He bath gas) of 2 to 3 torr. The majority (63%) of the peptide ion collision cross-sections correlate well with structures that are best described as charge-solvated globules, but a significant number of the peptide ions exhibit collision cross-sections that are significantly larger or smaller than the average, globular mobility-mass correlation. Of the peptide ions having larger than average collision cross-sections, approximately 71% are derived from trypsin digestion (C-terminal Arg or Lys residues) and most of the peptide ions that have smaller (than globular) collision cross-sections are derived from pepsin digestion (90%).

An efficient fluorescent protein-based multifunctional affinity purification approach in mammalian cells.

Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

Identification of SIN pathway targets reveals mechanisms of crosstalk between NDR kinase pathways.

The septum initiation network (SIN) regulates multiple functions during late mitosis to ensure successful completion of cytokinesis in Schizosaccharomyces pombe. One mechanism by which the SIN promotes cytokinesis is by inhibiting a competing polarity pathway called the MOR, which is required for initiation of polarized growth following completion of cytokinesis. Mutual antagonism between the two NDR kinase pathways, SIN and MOR, is required to coordinate cytoskeletal rearrangements during the mitosis-interphase transition. To determine how the SIN regulates the MOR pathway, we developed a proteomics approach that allowed us to identify multiple substrates of the SIN effector kinase Sid2, including the MOR pathway components Nak1 kinase and an associated protein, Sog2. We show that Sid2 phosphorylation of Nak1 causes removal of Nak1 from the spindle pole bodies, which may both relieve Nak1 inhibition of the SIN and block MOR signaling by preventing interaction of Nak1 with the scaffold protein Mor2. Because the SIN and MOR are conserved in mammalian cells (Hippo and Ndr1/2 pathways, respectively), this work may provide important insight into how the activities of these essential pathways are coordinated.

The kinesin-14 Klp2 is negatively regulated by the SIN for proper spindle elongation and telophase nuclear positioning.

In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end-directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized.

Predicting the fission yeast protein interaction network.

A systems-level understanding of biological processes and information flow requires the mapping of cellular component interactions, among which protein-protein interactions are particularly important. Fission yeast (Schizosaccharomyces pombe) is a valuable model organism for which no systematic protein-interaction data are available. We exploited gene and protein properties, global genome regulation datasets, and conservation of interactions between budding and fission yeast to predict fission yeast protein interactions in silico. We have extensively tested our method in three ways: first, by predicting with 70-80% accuracy a selected high-confidence test set; second, by recapitulating interactions between members of the well-characterized SAGA co-activator complex; and third, by verifying predicted interactions of the Cbf11 transcription factor using mass spectrometry of TAP-purified protein complexes. Given the importance of the pathway in cell physiology and human disease, we explore the predicted sub-networks centered on the Tor1/2 kinases. Moreover, we predict the histidine kinases Mak1/2/3 to be vital hubs in the fission yeast stress response network, and we suggest interactors of argonaute 1, the principal component of the siRNA-mediated gene silencing pathway, lost in budding yeast but preserved in S. pombe. Of the new high-quality interactions that were discovered after we started this work, 73% were found in our predictions. Even though any predicted interactome is imperfect, the protein network presented here can provide a valuable basis to explore biological processes and to guide wet-lab experiments in fission yeast and beyond. Our predicted protein interactions are freely available through PInt, an online resource on our website (www.bahlerlab.info/PInt).

N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast.

BACKGROUND: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. CONCLUSIONS/SIGNIFICANCE: This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.

Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

SIN-inhibitory phosphatase complex promotes Cdc11p dephosphorylation and propagates SIN asymmetry in fission yeast.

BACKGROUND: Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis. RESULTS: Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-inhibitory PP2A complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB. CONCLUSIONS: Because Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p dephosphorylation and the resulting recruitment of Byr4p are among the earliest steps in the establishment of SIN asymmetry.

Factors that influence helical preferences for singly charged gas-phase peptide ions: the effects of multiple potential charge-carrying sites.

Ion mobility-mass spectrometry is used to investigate the structure(s) of a series of model peptide [M + H](+) ions to better understand how intrinsic properties affect structure in low dielectric environments. The influence of peptide length, amino acid sequence, and composition on gas-phase structure is examined for a series of model peptides that have been previously studied in solution. Collision cross sections for the [M + H](+) ions of Ac-(AAKAA)(n)Y-NH(2) (n = 3-6) and Ac-Y(AEAAKA)(n)F-NH(2) (n = 2-5) are reported and correlated with candidate structures generated using molecular modeling techniques. The [M + H](+) ions of the AAKAA peptide series each exhibit a single, dominant ion mobility arrival time distribution (ATD) which correlates to partial helical structures, whereas the [M + H](+) ions of the AEAAKA ion series are composed of ATDs which correlate to charge-solvated globules (i.e., the charge is coordinated or solvated by polar peptide functional groups). These data raise numerous questions concerning intrinsic properties (amino acid sequence and composition as well as charge location) that dictate gas-phase peptide ion structure, which may reflect trends for peptide ion structure in low dielectric environments, such as transmembrane segments.