Family Of GTP-binding Proteins Research Articles

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Membrane trafficking alterations in breast cancer progression.

Breast cancer (BC) is the most common type of cancer in women, and remains one of the major causes of death in women worldwide. It is now well established that alterations in membrane trafficking are implicated in BC progression. Indeed, membrane trafficking pathways regulate BC cell proliferation, migration, invasion, and metastasis. The 22 members of the ADP-ribosylation factor (ARF) and the >60 members of the rat sarcoma (RAS)-related in brain (RAB) families of small GTP-binding proteins (GTPases), which belong to the RAS superfamily, are master regulators of membrane trafficking pathways. ARF-like (ARL) subfamily members are involved in various processes, including vesicle budding and cargo selection. Moreover, ARFs regulate cytoskeleton organization and signal transduction. RABs are key regulators of all steps of membrane trafficking. Interestingly, the activity and/or expression of some of these proteins is found dysregulated in BC. Here, we review how the processes regulated by ARFs and RABs are subverted in BC, including secretion/exocytosis, endocytosis/recycling, autophagy/lysosome trafficking, cytoskeleton dynamics, integrin-mediated signaling, among others. Thus, we provide a comprehensive overview of the roles played by ARF and RAB family members, as well as their regulators in BC progression, aiming to lay the foundation for future research in this field. This research should focus on further dissecting the molecular mechanisms regulated by ARFs and RABs that are subverted in BC, and exploring their use as therapeutic targets or prognostic markers.

Identification, characterization and functional analysis of Rap1 in response to bacterial infection in tilapia

Rap1 (Ras-related protein 1), as a member of the Ras-like small GTP binding protein family, involves in the regulation of cell life activities and mediates a variety of biological events including cell junctions, cell polarity, cell proliferation and differentiation by binding to GDP/GTP. In addition, Rap1 also plays an important role in innate immune response by regulating the adhesion, diffusion and migration of white blood cells. However, it is still unclear how Rap1 affects the innate immunity of teleost fish against bacterial infections. In this study, we amplified the Rap1 from tilapia and conducted the sequence analysis of Rap1 nucleotide and amino acid sequences, then characterized its tissue distribution and expression pattern in response to pathogen infection in tilapia for the first time, and explored the bacteriostatic and bacterial binding effects of Rap1. The results showed that the Rap1 possessed a whole open reading frame (ORF) with 558 bp, encoding 185 amino acids. The Rap1 protein was highly homologous with Rap1 from other species. Conserved domain analysis revealed that Rap1 contained five G box domains that mediated its binding to ornithine. Rap1 mRNA was widely expressed in tilapia tissues, and the highest level was found in spleen. Under Streptococcus agalactiae (S. agalactiae) infection, the expression of Rap1 was significantly up-regulated in head kidney and brain. In addition, the recombinant protein Rap1 hold the ability binding to S. agalactiae and inhibit the proliferation of S. agalactiae. In conclusion, this study not only provided a reference for exploring the function of Rap1 in teleost against pathogen infection, but also enriched the basic theoretical research of Rap1 function and antibacterial immune response in teleost.

Cdc42 couples septin recruitment to the axial landmark assembly via Axl2 in budding yeast.

Cell polarization generally occurs along a single axis that is directed by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae undergo polarized growth and oriented cell division in a spatial pattern by selecting a specific bud site. Haploid a or α cells bud in the axial pattern in response to a transient landmark that includes Bud3, Bud4, Axl1, and Axl2. Septins, a family of filament-forming GTP-binding proteins, are also involved in axial budding and recruited to an incipient bud site, but the mechanism of recruitment remains unclear. Here, we show that Axl2 interacts with Bud3 and the Cdc42 GTPase in its GTP-bound state. Axl2 also interacts with Cdc10, a septin subunit, promoting efficient recruitment of septins near the cell division site. Furthermore, a cdc42 mutant defective in the axial budding pattern at a semi-permissive temperature had a reduced interaction with Axl2 and compromised septin recruitment in the G1 phase. We thus propose that active Cdc42 brings Axl2 to the Bud3-Bud4 complex and that Axl2 then interacts with Cdc10, linking septin recruitment to the axial landmark.

Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule invitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.

Migration of Myogenic Cells Is Highly Influenced by Cytoskeletal Septin7

Septin7 as a unique member of the GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be essential in the formation of hetero-oligomeric septin complexes. As a cytoskeletal component, Septin7 is involved in many important cellular processes. However, its contribution in striated muscle physiology is poorly described. In skeletal muscle, a highly orchestrated process of migration is crucial in the development of functional fibers and in regeneration. Here, we describe the pronounced appearance of Septin7 filaments and a continuous change of Septin7 protein architecture during the migration of myogenic cells. In Septin7 knockdown C2C12 cultures, the basic parameters of migration are significantly different, and the intracellular calcium concentration change in migrating cells are lower compared to that of scrambled cultures. Using a plant cytokinin, forchlorfenuron, to dampen septin dynamics, the altered behavior of the migrating cells is described, where Septin7-depleted cells are more resistant to the treatment. These results indicate the functional relevance of Septin7 in the migration of myoblasts, implying its contribution to muscle myogenesis and regeneration.

Functional Characterization of Tomato ShROP7 in Regulating Resistance against Oidium neolycopersici.

ROPs (Rho-like GTPases from plants) are a unique family of small GTP-binding proteins in plants and play vital roles in numerous cellular processes, including growth and development, abiotic stress signaling, and plant defense. In the case of the latter, the role of ROPs as response regulators to obligate parasitism remains largely enigmatic. Herein, we isolated and identified ShROP7 and show that it plays a critical role in plant immune response to pathogen infection. Real-time quantitative PCR analysis revealed that the expression of ShROP7 was significantly increased during incompatible interactions. To establish its requirement for resistance, we demonstrate that virus-induced gene silencing (VIGS) of ShROP7 resulted in increased susceptibility of tomato to Oidium neolycopersici (On) Lanzhou strain (On-Lz). Downstream resistance signaling through H2O2 and the induction of the hypersensitive response (HR) in ShROP7-silenced plants were significantly reduced after inoculating with On-Lz. Taken together, with the identification of ShROP7-interacting candidates, including ShSOBIR1, we demonstrate that ShROP7 plays a positive regulatory role in tomato powdery mildew resistance.

Abstract 2681: Novel and selective inhibitors of KRASG12D

Abstract RAS proteins are family of small GTP-binding proteins that play a major role in regulating cellular responses such as proliferation, differentiation, migration, and cell growth. RAS proteins function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states, which relay cellular signals in response to extracellular stimuli. Aberrant RAS signaling plays a role in up to 30% of all human cancers, with the highest incidence occurring in carcinomas of the pancreas (63–90%), colon (36–50%), and lung (19–30%). Targeting the inactive, GDP bound KRASG12C has been a promising approach for generating novel anti RAS therapies, which is validated by the approval of AMG 510 (Lumakras) by the FDA. It is also known that KRASG12D also retains GTPase activity. Consequently, KRASG12D is one of the most important chemotherapeutic drug targets. KRASG12D is commonly observed in pancreatic cancer. Here we describe preclinical profile of a potent, selective, in-vivo tool compound, VRTX144. VRTX144 was potent in HPAC cell line, representing pancreatic adenocarcinoma, and a panel of KRASG12D mutated cell lines. It was found to be several folds less active in non-KRASG12D mutant cell lines. In vitro combination strategies with other targeted therapies such as EGFR, SHP2 and CDK4/6 inhibitor is also being investigated. VRTX144 exhibited low propensity for metabolism in microsomes, and has high protein bound. VRTX144 was found to have no adverse effect in hERG upto 10 uM. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Novel and selective inhibitors of KRASG12D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2681.

Molecular characterization and expression analysis of septin gene family and phagocytic function of recombinant septin 2, 3 and 8 of starry flounder (Platichthys stellatus)

Septin is an evolutionarily conserved family of GTP-binding proteins. Septins are known to be involved in a variety of cellular processes, including cell division, chromosome separation, cell polarity, motility, membrane dynamics, exocytosis, apoptosis, phagocytosis, DNA damage responses, and other immune responses. In this study, the sequences of the septin gene family of starry flounder were obtained using NGS sequencing, and the integrity of the sequences was verified through cloning and sequencing. At first, the amino acid sequence was annotated using the cDNA sequence, and then, the gene sequence was verified through multiple sequence alignment and phylogenetic analyses using the related conserved sequences. The septin gene family was classified into three subgroups based on the phylogenetic analysis. High conservation within the domain and homology between the genes reported in different species were confirmed. The expression level of septin gene family mRNA in each tissue of healthy starry flounder was evaluated to confirm the tissue- and gene-specific expression levels. Additionally, as a result of the analysis of mRNA expression after simulated pathogen infection, significant expression changes and characteristics were confirmed upon infection with bacteria (Streptococcus parauberis PH0710) and virus (VHSV). Based on the current results and that of previous studies, to confirm the immunological function, Septin 2, 3, and 8 were produced as recombinant proteins based on the amino acid sequences, and their role in phagocytosis was further investigated. The results of this study indicate that septin gene family plays a complex and crucial role in the host immune response to pathogens of starry flounder.

Novel mechanisms in phosphoinositide turnover

The hydrolysis of phosphatidylinositol 4,5‐ bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K,dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2synthesis during phototransduction. The activity of dPIP5KLin vitrois enhanced by depletion of PNUT, a non‐redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of dPIP5KL rescues the effect of pnut depletion on the light response and PI(4,5)P2resynthesis during PLC signalling. Thus, our work defines a septin 7 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

Septin 2 interacts with dengue virus replication complex proteins and participates in virus replication in mosquito cells

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

EP190: A third patient with RAP1B-related syndromic thrombocytopenia and novel clinical findings

RAP1B is a member of the RAS family of small GTP binding proteins (GTPases), activated in many signal transduction pathways regulating numerous cellular processes including proliferation, adhesion, and migration. RAP1B-related syndromic thrombocytopenia is an ultra-rare genetic disorder characterized by hematologic abnormalities, neurodevelopmental delays, and congenital birth defects. Initial report of a germline RAP1B variant described a patient with a Kabuki syndrome-like phenotype due to RAP1B loss-of-function.

The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin.

Septins are a family of filament-forming GTP-binding proteins that regulate fundamental cellular activities, such as cytokinesis and cell polarity. In general, septin filaments function as barriers and scaffolds on the cell cortex. However, little is known about the mechanism that governs the recruitment and localization of the septin complex to the cell cortex. Here, we identified the Cdc42 GTPase-activating protein Rga6 as a key protein involved in promoting the localization of the septin complex to the cell cortex in the fission yeast Schizosaccharomyces pombe. Rga6 interacts with the septin complex and partially colocalizes with the septin complex on the cell cortex. Live-cell microscopy analysis further showed septin enrichment at the cortical regions adjacent to the growing cell tip. The septin enrichment likely plays a crucial role in confining active Cdc42 to the growing cell tip. Hence, our findings support a model whereby Rga6 regulates polarized cell growth partly through promoting targeted localization of the septin complex on the cell cortex. This article has an associated First Person interview with the first author of the paper.

ADP-Ribosylation Factor Family of Small GTP-Binding Proteins: Their Membrane Recruitment, Activation, Crosstalk and Functions.

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide binding proteins play critical roles in various cellular processes, especially in regulating the secretory, and endocytic pathways. The fidelity of intracellular vesicular trafficking depends on proper activations and precise subcellular distributions of ARF family proteins regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here we review recent progress in understanding the membrane recruitment, activation, crosstalk, and functions of ARF family proteins.

Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.

Multiple Omics Analysis of the Rac3 Roles in Different Types of Human Cancer

Rac Family Small GTPase 3 (Rac3) is a member of the Rho family of small GTP-binding proteins which play critical roles in the occurrence, progression, and metastasis of various tumors. Nevertheless, previous studies have focused on a single type of human cancer, so that the roles of Rac3 in different cancer types have not been sufficiently clarified. With the progress of biological detection and bioinformatics technology, the emerging cancer genomics databases make it possible to perform a pan-cancer analysis. Therefore, for the first time, we performed multiple omics analysis to investigate the roles of Rac3 in differential expression, survival prognosis, mutative status, DNA methylation, functions, immune infiltration, and immunotherapy across 33 human cancer types. We found that Rac3 expression was abnormal in 17 cancer types and significantly different in both molecular and immune subtypes of 10 cancers (p<0.05). These Rac3 expression dysregulations in cancer tissues significantly affected their corresponding survival prognosis. Our results also indicated that genetic alterations of Rac3 occurred in 27 cancer types and were significantly associated with prognosis. Moreover, methylation of Rac3 was associated with dysfunctional T-cell phenotypes and affected the prognosis of the brain, melanoma, and breast tumors, and Rac3 Copy Number Alterations (CNA) might affect the infiltration levels of different immune cells in nine cancers. Rac3 and Rac3-related genes were enriched in the axon guidance, actin cytoskeleton regulation, and neurotrophin signaling pathway, and involved the regulation of cellular localization and actin cytoskeleton organization, and small GTPase mediated signal transduction. Then, we further explored the correlations between Rac3 expression and 25 immune cells and cancer-associated fibroblasts across 33 human cancers. Furthermore, we found that Rac3 achieved higher predictive power (AUC>0.5) than three standardized biomarkers of tumor immune responses and was associated with cytotoxic T-cell levels (CTLs) and the risk of immunotherapeutic responses in three cancer types. Collectively, our study contributes to a comprehensive and integrative understanding of the oncogenic roles of Rac3 across human cancers and offers a new clue for treatment strategies.

Association Mapping and Functional Analysis of Rice Cold Tolerance QTLs at the Bud Burst Stage

Cold tolerance at the bud burst stage (CTB) is a key trait for direct-seeded rice. Although quantitative trait loci (QTL) affecting CTB in rice have been mapped using traditional linkage mapping and genome-wide association study (GWAS) methods, the underlying genes remain unknown. In this study, we evaluated the CTB phenotype of 339 cultivars in the Rice Diversity Panel II (RDP II) collection. GWAS identified four QTLs associated with CTB (qCTBs), distributed on chromosomes 1–3. Among them, qCTB-1-1 overlaps with Osa-miR319b, a known cold tolerance micro RNA gene. The other three qCTBs have not been reported. In addition, we characterised the candidate gene OsRab11C1 for qCTB-1-2 that encodes a Rab protein belonging to the small GTP-binding protein family. Overexpression of OsRab11C1 significantly reduced CTB, while gene knockout elevated CTB as well as cold tolerance at the seedling stage, suggesting that OsRab11C1 negatively regulates rice cold tolerance. Molecular analysis revealed that OsRab11C1 modulates cold tolerance by suppressing the abscisic acid signalling pathway and proline biosynthesis. Using RDP II and GWAS, we identified four qCTBs that are involved in CTB and determined the function of the candidate gene OsRab11C1 in cold tolerance. Our results demonstrate that OsRab11C1 is a negative regulator of cold tolerance and knocking out of the gene by genome-editing may provide enhanced cold tolerance in rice.

Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells.

Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary significantly in their N-terminal extensions. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (e.g., nuclei, centrosomes, cilia) and functions such as cell signalling and ubiquitination. The interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear, signalling, and ubiquitinating proteins. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.

A nuclear proteome localization screen reveals the exquisite specificity of Gpn2 in RNA polymerase biogenesis

ABSTRACT The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.