The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics.

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Functional role of autophagy-mediated proteome remodeling in cell survival signaling and innate immunity.

Ras-driven cancer cells upregulate basal autophagy that degrades and recycles intracellular proteins and organelles. Autophagy-mediated proteome degradation provides free amino acids to support metabolism and macromolecular synthesis, which confers a survival advantage in starvation and promotes tumorigenesis. While the degradation of isolated protein substrates by autophagy has been implicated in controlling cellular function, the extent and specificity by which autophagy remodels the cellular proteome and the underlying functional consequences were unknown. Here we compared the global proteome of autophagy-functional and -deficient Ras-driven cancer cells, finding that autophagy affects the majority of the proteome yet is highly selective. While levels of vesicle trafficking proteins important for autophagy are preserved during starvation-induced autophagy, deleterious inflammatory response pathway components are eliminated even under basal conditions, preventing cytokine-induced paracrine cell death. This reveals the global, functional impact of autophagy-mediated proteome remodeling on cell survival and identifies critical autophagy substrates that mediate this process.

Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation.

Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19's substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery.

Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate.

Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). PGAM1 needs to be histidine phosphorylated to become catalytically active. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is controlling glycolytic intermediate levels and thereby serine biosynthetic flux.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms.

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of ß-barrel proteins.

RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of ß-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all ß-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the ß-barrel assembly machine.

Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

BACKGROUND: Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown. RESULTS: Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction. CONCLUSIONS: These results identify cryptic protein interaction motifs in the cytoplasmic domain of MHCI. In so doing, they suggest that the cytoplasmic domain of MHCI could participate in previously unsuspected PDZ mediated protein-protein interactions at neuronal as well as immunological synapses.

Impact of the adenoviral E4 Orf3 protein on the activity and posttranslational modification of p53.

UNLABELLED: Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076-1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE: The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55-kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the posttranslational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences that are deleterious for viral reproduction, such as apoptosis or cell cycle arrest.

Extensive post-translational modification of active and inactivated forms of endogenous p53.

The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.

Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

A pan-specific antibody for direct detection of protein histidine phosphorylation.

Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.

Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a "PDH-like" enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.

A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism.

Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC-MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC-MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.

ChelomEx: Isotope-assisted discovery of metal chelates in complex media using high-resolution LC-MS.

Chelating agents can control the speciation and reactivity of trace metals in biological, environmental, and laboratory-derived media. A large number of trace metals (including Fe, Cu, Zn, Hg, and others) show characteristic isotopic fingerprints that can be exploited for the discovery of known and unknown organic metal complexes and related chelating ligands in very complex sample matrices using high-resolution liquid chromatography mass spectrometry (LC-MS). However, there is currently no free open-source software available for this purpose. We present a novel software tool, ChelomEx, which identifies isotope pattern-matched chromatographic features associated with metal complexes along with free ligands and other related adducts in high-resolution LC-MS data. High sensitivity and exclusion of false positives are achieved by evaluation of the chromatographic coherence of the isotope pattern within chromatographic features, which we demonstrate through the analysis of bacterial culture media. A built-in graphical user interface and compound library aid in identification and efficient evaluation of results. ChelomEx is implemented in MatLab. The source code, binaries for MS Windows and MAC OS X as well as test LC-MS data are available for download at SourceForge ( http://sourceforge.net/projects/chelomex ).

Gut microbial ß-glucuronidases influence endobiotic homeostasis and are modulated by diverse therapeutics.

Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.

Novel phosphorylation sites in the S. cerevisiae Cdc13 protein reveal new targets for telomere length regulation.

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. Multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics.