GFP Fusion Constructs Research Articles

A unique mutation in PIN-FORMED1 and a genetic pathway for reduced sensitivity of Arabidopsis roots to N-1-naphthylphthalamic acid.

The small chemical N-1-naphthylphthalamic acid (NPA) has long been used as a polar auxin transport inhibitor. Recent biochemical and structural investigations have revealed that this molecule competes with the auxin IAA (indole-3-acetic acid) inside the PIN-FORMED auxin efflux carriers. However, the existence of any mutations in PIN family proteins capable of uncoupling the docking of IAA from NPA remains unclear. We report that Arabidopsis thaliana seedlings overexpressing SMALL AUXIN UP RNA 41 were hypersensitive to NPA-induced root elongation inhibition. We mutagenized this line to improve the genetic screening efficiency for NPA hyposensitivity mutants. Using bulked segregation analysis and mapping-by-sequencing assessment of these mutants, we identified a core genetic pathway for NPA-induced root elongation inhibition, including genes required for auxin biosynthesis, transportation, and signaling. To evaluate specific changes of auxin signaling activity in mutant roots before and after NPA treatment, the DR5::GFP/DR5::YFP markers were introduced and observed. Most importantly, we discovered a unique mutation in the PIN1 protein, substituting a proline residue with leucine at position 584, leading to a loss of NPA sensitivity while keeping the auxin efflux capacity. Transforming the null mutant pin1-201 with the PIN1::PIN1P584L -GFP fusion construct rescued the PIN1 function and provided NPA hyposensitivity. The proline residue is predicted to be adjacent to a hinge in the middle region of the ninth transmembrane helix of PIN1 and is conserved from moss to higher plants. Our work may bring new insights into the engineering of NPA-resistant PINs for auxin biology studies.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

Identification of a stretch of four discontinuous amino acids involved in regulating kinase activity of IGF1R.

IGF1R is pursued as a therapeutic target because of its abnormal expression in various cancers. Recently, we reported the presence of a putative allosteric inhibitor binding pocket in IGF1R that could be exploited for developing novel anti-cancer agents. In this study, we examined the role of nine highly conserved residues surrounding this binding pocket, with the aim of screening compound libraries in order to develop small-molecule allosteric inhibitors of IGF1R. We generated GFP fusion constructs of these mutants to analyze their impact on subcellular localization, kinase activity and downstream signaling of IGF1R. K1055H and E1056G were seen to completely abrogate the kinase activity of IGF1R, whereas R1064K and L1065A were seen to significantly reduce IGF1R kinase activity. During molecular dynamics analysis, various structural and conformational changes were observed in different conserved regions of mutant proteins, particularly in the activation loop, compromising the kinase activity of IGF1R. These results show that a stretch of four discontinuous residues within this newly identified binding pocket is critical for the kinase activity and structural integrity of IGF1R. This article has an associated First Person interview with the first author of the paper.

Siamese Cat Tyrosinase has Enhanced Proteasome Degradation and Increased Cellular Aggregation

Aim: Siamese cats are a notable example of a temperature-sensitive partial albinism phenotype. The signature color-pointing pattern is the result of an amino acid substitution - G302R - in the cysteine-rich domain of feline tyrosinase. The precise mechanism for the loss of tyrosinase enzyme activity due to this mutation is unknown. Objective: We have used a cellular biology approach to unraveling the relationships between feline coloration, behavior and increased risk for feline cognitive dysfunction syndrome. GFP-fusion constructs of wild type domestic short hair tyrosinase and Siamese (G302R) tyrosinase were generated to study cellular trafficking, degradation and the propensity for cellular aggregation. Data Description: C-terminal GFP G302R expression has reduced Golgi localization, increased cytosolic fractions with reduced calnexin co-localization. N-terminal GFP constructs were retained in the ER, with little to no Golgi associated forms. C-terminal and N-terminal GFP G302R TYR is observed to have increased high molecular weight aggregation following proteasome inhibition.

Metal tolerance protein family members are involved in Mn homeostasis through internal compartmentation and exocytosis in Brassica napus

Mineral elements, including manganese (Mn), are indispensable for plant growth and development. However, plants cultivated on acidic soils are vulnerable to Mn2+ toxicity due to a prevalently phytotoxic level of Mn2+. Metal tolerance proteins (MTPs) were demonstrated to be essential for metal homeostasis and tolerance in different plant species. In this study, we present the functional characteristics of BnMTP8 and BnMTP9 from Brassica napus. The expression level of BnMTP8 was most prominent in rapeseed roots and was only markedly up-regulated in shoots by excess Mn2+. In contrast, BnMTP9 was most abundant in shoots. Expression of BnMTP8 and BnMTP9 in Saccharomyces cerevisiae compensated the Mn2+-hypersensitivity of Δpmr1 by increasing metal sequestration and efflux, respectively. In addition, heteroexpression of BnMTP8 restored Mn2+ tolerance of Arabidopsis mtp8 mutants and increased Mn2+ accumulation in tissues and leaf vacuoles. Expression of BnMTP9 in Arabidopsis mtp11 mutants restored their growth and reduced Mn2+ concentrations in mesophyll protoplasts and tissues upon elevated levels of Mn2+. Transient expression of GFP-fusion constructs in protoplasts showed that BnMTP8 and BnMTP9 localize to pre-vacuolar compartments and the trans-Golgi network, respectively. However, high extracellular Mn2+ in Arabidopsis root cells triggered BnMTP8-GFP relocation to the tonoplast and BnMTP9-GFP relocation to the plasma membrane, suggesting that these two proteins are involved in vacuolar Mn2+ sequestration and cytosolic Mn2+ export, respectively. Taken together, these findings indicate that BnMTP8 and BnMTP9 load Mn2+ into vesicles for subsequent delivery to the vacuole or secretion into extracellular spaces, respectively, thereby cooperating to maintain Mn2+ homeostasis in the roots and shoots of rapeseed.

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley.

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

Speckled 100kDa gene in pigs: Alternative splicing, subcellular localization, and response to interferon-α stimulation.

Speckled 100kDa (Sp100) plays an important role in the antiviral immune response, however, little is known about porcine Sp100. In this study, porcine Sp100 was cloned and its response to interferon (IFN) α was identified. We obtained the cDNA (V1) of the gene, SP100, and seven alternative splicing variants (V2-8). Isoform V1 encoded a 386 amino acid protein and contained a homogeneously-staining region (HSR) domain. Isoforms V3, 4, 6 and 7 were deletion/insertion variants and contained HSR domain as V1. The splicing of porcine SP100 was very complicated and many transcripts existed as revealed by cloning and minigene analyses. Using GFP-fusion constructs isoforms V1, 3, 4, 6 and 7 were localized to nucleus and the nuclear localization signal was identified as PSNRKRR at positions 331-337 of V1. Porcine SP100 was unevenly distributed in all tissues studied and differentially expressed between pigs with different disease-resistance/susceptibilities. Porcine SP100 was strongly increased by IFNα due to the existence of an IFN-stimulated response element in the promoter. A single nucleotide-70A>C polymorphism enhanced promoter activity. The results provided the basis for determining the role of Sp100 in antiviral responses and may assist in breeding pigs with high disease-resistance.

Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent β-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety ‘Siberia’

Lilies are a commercially significant cut flower worldwide due not only to their elegant shape but also to their appealing scent. Among Lilium varieties, Lilium 'Siberia' is a cultivar that is prominent and highly favored by consumers due to its snowy white color and strong floral scent. Here, two terpene synthase genes (LoTPS2 and LoTPS4) that are responsible for floral scent production in Lilium 'Siberia' were cloned and functionally characterized. Recombinant LoTPS2 specifically catalyzed the formation of (E, E)-α-farnesene from FPP. Recombinant LoTPS4 is a multiproduct enzyme that produces D-limonene and β-myrcene as major volatile compounds and β-phellandrene, (+)-4-carene and 3-carene as minor products from GPP. Furthermore, LoTPS4 generates trans-α-bergamotene as a major product and di-epi-α-cedrene, α-cubebene and (E)-β-farnesene as minor compounds from FPP. Subcellular localization analysis using GFP fusion constructs revealed that LoTPS2 was localized in the cytosol, whereas LoTPS4 was localized in plastids. Real-time PCR analysis showed that LoTPS2 was highly expressed in the petals and sepals of the flower, while LoTPS4 was highly expressed in the filament of the flower. Moreover, mechanical wounding of flowers revealed that LoTPS2 showed a strong response to wounding via a rapid increase in its mRNA transcript level. Our results will assist scientists in exploring the molecular mechanisms of terpene biosynthesis in this species and will provide new insight into the biotechnological modification of the floral bouquet in Lilium.

A PAS-Containing Histidine Kinase is Required for Conidiation, Appressorium Formation, and Disease Development in the Rice Blast Fungus, Magnaporthe oryzae

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.).

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

Protracted and asynchronous accumulation of PSD95-family MAGUKs during maturation of nascent dendritic spines.

The formation and stabilization of new dendritic spines is a key component of the experience-dependent neural circuit plasticity that supports learning, but the molecular maturation of nascent spines remains largely unexplored. The PSD95-family of membrane-associated guanylate kinases (PSD-MAGUKs), most notably PSD95, has a demonstrated role in promoting spine stability. However, nascent spines contain low levels of PSD95, suggesting that other members of the PSD-MAGUK family might act to stabilize nascent spines in the early stages of spiny synapse formation. Here, we used GFP-fusion constructs to quantitatively define the molecular composition of new spines, focusing on the PSD-MAGUK family. We found that PSD95 levels in new spines were as low as those previously associated with rapid subsequent spine elimination, and new spines did not achieve mature levels of PSD95 until between 12 and 20 h following new spine identification. Surprisingly, we found that the PSD-MAGUKs PSD93, SAP97, and SAP102 were also substantially less enriched in new spines. However, they accumulated in new spines more quickly than PSD95: SAP102 enriched to mature levels within 3 h, SAP97 and PSD93 enriched gradually over the course of 6 h. Intriguingly, when we restricted our analysis to only those new spines that persisted, SAP97 was the only PSD-MAGUK already present at mature levels in persistent new spines when first identified. Our findings uncover a key structural difference between nascent and mature spines, and suggest a mechanism for the stabilization of nascent spines through the sequential arrival of PSD-MAGUKs. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1161-1174, 2017.

Regulatory Characteristics of Bacillus pumilus Protease Promoters

Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5' regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3-19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250bp in length, respectively. Promoter analysis of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis.

Molecular characterization of Sp110 gene in pigs.

Speckled 110kDa (Sp110) plays an important role in infectious diseases, as revealed by studies in humans. However, little is known regarding porcine Sp110. To elucidate its potential role in porcine resistance to viral diseases, here, the complete coding sequence of porcine Sp110 gene and its 26 alternatively spliced isoforms were isolated using reverse transcription (RT)-polymerase chain reaction (PCR), and another seven splicing patterns were obtained using a minigene construct. Subcellular distribution of 11 representative isoforms was characterized in PK-15 cells transiently transfected with their respective GFP fusion constructs, and only isoforms (R and V) bearing all functional domains were localized in nucleus, indicating all the other isoforms lose normal functions of Sp110 owing to alternative splicing. Real-time quantitative PCR and competitive RT-PCR showed that both isoforms R and V had similar tissue expression profile, half-life and response to poly(I:C), a synthetic analog of viral double-stranded RNA, while the longer one (isoform R) was transcribed at a higher level. The results indicated that porcine Sp110 has a role in viral infection and that isoform R is the dominant active form. Overall the data provide potential resource for molecular breeding of pig resistant to diseases and contributes to breeding pigs resistant to viral infection.

Liposome-Based in Vitro Biosynthesis of Two-Pore Domain Potassium Channels for Functional Studies

The Two-pore domain potassium channels (K2P) function as regulators of membrane resting potential and cellular electrical excitability. The family has 15 homo- and heterodimeric members, most of which can be characterized by electrophysiological recordings of transfected cells and RNA-injected Xenopus oocytes.However, drawbacks of heterologuous expression arise from ungovernable cellular regulation events, unknown membrane composition, or lack of wildtype currents at the plasma membrane. Alternative methods demand recombinant membrane protein production, which is still difficult.We report the in vitro biosynthesis of several K2P channels as well as mutant and GFP-fusion constructs in a lipid-containing wheat-germ cell-free system.We show that Giant Unilamellar Vesicles (GUV) as well as Small Unilamellar Vesicles (SUV) of defined lipid composition can be included in the cell-free reaction to promote production of full-length channel proteins without addition of detergents. Channel insertion and orientation in the GUV lipid membrane was visualized by fluorescence microscopy of GFP-tagged fusion proteins and a proteolytic assay. Channel containing GUV were probed by patch-clamp methods, while SUV were preferred for planar lipid bilayer experiments. Our results indicate that K2P channels are formed in a functional state.Liposome-based cell-free synthesis provides the opportunity of obtaining ion channels in artificial membrane environment to study lipid regulation effects or mechanosensitivity exclusive of endogenuous regulatory or accessory proteins. and coexpression experiments could reveal interaction partners. Furthermore, patch clamp techniques can be extended to channels of possibly intracellular location and could help to understand the role of K2P family members so far referred to as “silent”. As synthesis is accessible for reagent addition, site-specific labeling by fluorophores, cysteine reactive agents or non-natural amino acids can readily be achieved and might prove beneficial in studying structural changes in gating events.

The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans.

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.

Riboflavin transporter-2 (rft-2) of Caenorhabditis elegans: Adaptive and developmental regulation.

Riboflavin transporters (rft-1 and rft-2), orthologous to human riboflavin transporter-3 (hRVFT-3), are identified and characterized in Caenorhabditis elegans. However, studies pertaining to functional contribution of rft-2 in maintaining body homeostatic riboflavin levels and its regulation are very limited. In this study, the expression pattern of rft-2 at different life stages of C. elegans was studied through real-time PCR, and found to be consistent from larval to adult stages that demonstrate its involvement in maintaining the body homeostatic riboflavin levels at whole animal level all through its life. A possible regulation of rft-2 expression at mRNA levels at whole animal was studied after adaptation to low and high concentrations of riboflavin. Abundance of rft-2 transcript was upregulated in riboflavin-deficient conditions (10 nM), while it was downregulated with riboflavin-supplemented conditions (2 mM) as compared with control (10 meu M). Further, the 5'-regulatory region of the rft-2 gene was cloned, and transgenic nematodes expressing transcriptional rft-2 promoter::GFP fusion constructs were generated. The expression of rft-2 was found to be adaptively regulated in vivo when transgenic worms were maintained under different extracellular riboflavin levels, which was also mediated partly via changes in the rft-2 levels that directs towards the possible involvement of transcriptional regulatory events.

Towards multiplexed protein–protein interaction analysis using protein tag-specific nanobodies

Dynamic protein–protein interactions (PPIs) are an integral part of cellular processes. The discovery of modulators that disrupt or stabilize such interactions is highly important to understand PPIs and address correlating diseases. Bead-based protein assays analyzing PPIs between bait- and prey-proteins exemplify emerging methodologies. To date, most studies employ purified bait-proteins from bacteria. Such proteins are of limited use as they do not undergo eukaryotic folding and lack posttranslational modifications.Here, we present a novel method to generate bead-based protein arrays combining μ-scale purification of bait-proteins combined with site-directed immobilization. First, we express individual bait-proteins as GST- or GFP-fusion constructs in bacterial and mammalian cells. Next, we purify and immobilize these bait-proteins from crude lysates using high affinity tag-specific nanobodies coupled to color-coded beads. Finally, we combined those bait-coupled beads in a protein-array for miniaturized multiplexed GST- and GFP pulldown studies. In a proof-of-principle we study dynamic changes of the endogenous prey-protein β-catenin following proteasomal inhibition or signaling pathway perturbation.Our strategy enables a fast isolation of highly pure and stable bait-proteins derived from small-scale expression cultures. We propose that this approach enables the generation of bead-based protein arrays comprising hundreds of bait-proteins from different expression systems to study complex PPIs. Biological significanceProtein arrays and multiplexed sandwich immunoassays, are widely applied to study protein–protein interaction or to investigate the signaling status of stimulated cells. This study describes for the first time the application of tag-specific nanobodies for site directed immobilization of bait-proteins from different expression systems to generate bead based protein arrays. The analysis of the Wnt-pathway activation by multiplexed μ-scale pulldowns demonstrated the advantages of eukaryotic expression systems regarding the stability and binding properties of individual bait proteins.This article is part of a Special Issue entitled: HUPO 2014.

The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility.

Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219). We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf =69μm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5μm/min vs. 6.3μm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum

The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfμ1) mediated protein trafficking in P. falciparum. In trophozoites Pfμ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfμ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfμ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfμ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfμ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.