Integration of protein binding interfaces and abundance data reveals evolutionary pressures in protein networks

Networks of protein-protein interactions have received considerable interest in the past two decades for their insights about protein function and evolution. Traditionally, these networks only map the functional partners of proteins; they lack further levels of data such as binding affinity, allosteric regulation, competitive vs noncompetitive binding, and protein abundance. Recent experiments have made such data on a network-wide scale available, and in this thesis I integrate two extra layers of data in particular: the binding sites that proteins use to interact with their partners, and the abundance or “copy numbers” of the proteins. By analyzing the networks for the clathrin-mediated endocytosis (CME) system in yeast and the ErbB signaling pathway in humans, I find that this extra data reveals new insights about the evolution of protein networks. The structure of the binding site or interface interaction network (IIN) is optimized to allow higher binding specificity; that is, a high gap in strength between functional binding and nonfunctional mis-binding. This strongly implies that mis-binding is an evolutionary error-load constraint shaping protein network structure. Another method to limit mis-binding is to balance protein copy numbers so that there are no “leftover” proteins available for mis-binding. By developing a new method to quantify balance in IINs, I show that the CME network is significantly balanced when compared to randomly sampled sets of copy numbers. Furthermore, IINs with a biologically realistic structure produce less mis-binding under balanced concentrations, when compared to random networks, but more mis-binding under unbalanced concentrations. This implies strong pressure for copy number balance and that any imbalance should occur for functional reasons. I thus explore some functional consequences of imbalance by constructing dynamic models of two poorly balanced subnetworks of the larger CME network. In general, I find that balanced copy numbers provide higher protein complex yield (number of complete complexes), but imbalance may allow cells to “bottleneck” a functional process, effectively turning complex formation on or off via spatial localization of subunits. Finally, I find that strongly binding proteins are more likely to be balanced, as these “sticky” proteins would be more likely to engage in mid-binding otherwise

Mitotic Exit: Thresholds and Targets

Cyclin dependent kinases (CDKs) are at the heart of the cell cycle. Throughout the cycle, these complexes modify many proteins, changing various aspects of their regulation (stability, localization, etc.). As cells exit mitosis, the CDK that has driven many of the cell cycle processes is inhibited and degraded, allowing many of the kinase substrates to return to their unphosphorylated state. This assures that each subsequent cell cycle is begun in the same naïve state, again ready for CDK-dependent regulation. The studies in this thesis focus on two mechanisms by which this restoration is accomplished in the budding yeast, Saccharomyces cerevisiae: (1) a transcriptional program that transcribes many of the genes required for physically dividing the mother and daughter cells and beginning the next round of cell division and (2) a phosphatase that specifically removes the phosphates from sites modified by CDK during exit from mitosis. Two transcription factors, Swi5 and Ace2, transcribe many of the genes required for physically dividing the mother and daughter cells and beginning the next round of cell division. Previously our lab has shown that locking mitotic cyclin levels, by inducing transcription of an undegradable form of the protein, causes dose-dependent delays in different cell cycle events. The first chapter addresses the contribution of the transcriptional program to this phenomenon. Interestingly, in these cells where mitotic cyclin levels were sustained, deletion of the transcription factor Swi5 increases the mitotic cyclin inhibition, specifically as it relates to budding and cytokinesis. Importantly, when phosphorylated by CDK, Swi5 is excluded from the nucleus, so in the second chapter, we investigate its localization when mitotic cyclin levels are locked. Swi5 still enters the nucleus. In fact in some cells, Swi5 enters the nucleus several times before the cell cycle advances. Given previous studies from our lab showing that the release of Cdc14 phosphatase also oscillates under these conditions, the reentry of Swi5 may support a model that a kinase/phosphatase balance allows cell cycle progression in these cells. All this suggests that Swi5 promotes the transcription of genes important for promoting cytokinesis and budding despite high mitotic cyclin levels. In the third chapter, we begin to assess the contribution of specific targets of the mitotic exit transcriptional program to the mitotic cyclin-dependent regulation of specific cell cycle events. Finally, Cdc14, a phosphatase that removes the phosphate groups added by CDKs, is sequestered for most of the cell cycle but released from the nucleolus during the end of mitosis. In the fourth chapter, we examine the physiological relevance of these dephosphorylation events on novel targets of the Cdc14 phosphatase

The Genetic Profiles of TIF1 and TIF2 Duplicate Genes

As one of the key steps in protein synthesis, translation initiation is subjected to multi-level regulation which is achieved via diverse mechanisms. The cell adjusts protein synthesis accordingly to its status and environment. The degree of contribution of the processes involved in the regulation of translation initiation is still poorly understood. The first part of this study focuses on identifying mechanisms of regulation in a translationally deficient yeast system, impaired by the loss of one or the other of the TIF1/2 duplicate genes, which together code for the eukaryotic initiation factor 4A (eIF4A). A major finding of this research is related to the functional competences associated with the two duplicate members of the gene pair. Although the genetic profile associated with TIF1 highlights a connection with transcriptional process, the majority of transcription-translation inter-talk is allocated with TIF2, along with a dense network of genetic interactions surrounding the SAGA complex. TIF2 is also the only paralog devoted to interactions with a substantial group of functionally related genes involved in early meiotic gene expression. Protein degradation in the global control of protein synthesis represents a fundamental process and accounts for diverse points of control, in particular through ubiquitination/deubiquitination. This research concludes that functional turnover of proteins and the translation/transcription inter-talk emerges as the most significant contributors to the sophistically regulated translational regulation, The second part of this study aims to determine the extent of similarity and divergence between the TIF1 and TIF2 paralogs. Growth of their individual deletion strains was challenged under different chemical and environmental conditions with the intent to explore the relative contributions of each duplicate in response to an extend range of perturbations. The pair of duplicates appeared convincingly robust in coping with these adversities under disparate cellular contexts, thus suggesting a highly conserved and backed-up genetic network. One of the primary treatments made use of lithium, a condition which was hoped to help, along with furthering our understanding of the TIF1 and TIF2 networks, in formulating an explanation on how augmented translation initiation overcomes lithium toxicity. Although this approach did not return results that could be used to address this unresolved topic, evaluation of genetic inhibition and suppression was highly instructive regarding the mechanisms of response triggered upon lithium/galactose stress. Regulation and synchronization of basic cellular processes were affected: emphasis brought on aspects of cell communication highlighted mechanisms articulated by kinase enzymes and the importance of repression of cell cycle progression in control of protein synthesis. Data from the screen also indicated the stress that combined lithium/galactose treatment places on central metabolic pathways, for instance those between the Leloir, gluconeogenesis, and trehalose pathways

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

The role of VAMP proteins in K+ channel regulation

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis and pathogen defense. A subset of SNAREs, including the Arabidopsis SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K+ channels and to moderate their gating at the plasma membrane. R-SNAREs, also known as vesicle-associated membrane proteins (VAMPs), are most commonly associated with trafficking vesicles. In guard cells of Arabidopsis, ABA-dependent stomatal closure was inhibited when the expression of VAMP71 family genes were suppressed by an antisense VAMP711 construct. Such results reveal that VAMPs play an important role in plant response to stress, especially in the regulation of stomatal closure under ABA treatment. Two R-SNAREs, VAMP721 and VAMP722, are known to assemble in SNARE core complexes with SYP121 and with its closest homolog SYP122, which raises the question whether the channel interaction might extend to the R-SNAREs leading to VAMP regulating channel gating. To answer this question, I investigated the interaction between the VAMP7 proteins with KC1 and KAT1 K+ channels by mating based Split-Ubiquitin System (mbSUS) assay, and verified these interactions by ratiometric bimolecular fluorescence complementation (rBiFC) assay. I found VAMP721 and VAMP722, but not VAMP723, interacted with the channels. The selective binding was associated with the VAMP longin domain, notably with Tyr57. What is the effect of the VAMP-K+ channel interaction on transmembrane ion transport and vesicle traffic? I found VAMP721 affected K+ channel gating and suppressed the K+ current within the physiological voltage range by electrophysiological analysis in Xenopus oocytes and in VAMP over-expression wild type Arabidopsis. The effect of VAMP721 on K+ channel regulation was opposite to the action of SYP121 on K+ channel. From localization, interaction and electrophysiological studies, I was able to show that Tyr57 is a key residue required both for VAMP-dependent gating of the K channels and for localization of the VAMPs at the plasma membrane. For vesicle traffic, I found overexpression of full length VAMP721 in Arabidopsis seedlings blocked the secretion of secYFP while coexpressing with KC1 K+ channel rescued the traffic block, demonstrating a potential action of VAMP-K+ channel interaction on secretory traffic. These results add to understanding the interaction between SNARE and K+ channels that link membrane traffic with osmotically active solute transport in the plant