Jens Pfannstiel

TREX is a conserved complex coupling transcription with messenger RNA export.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)+ RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX (‘transcription/export’) complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.

Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling.

The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N‐terminal domain of Ndc80 (Ndc801−257), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc801−257 in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild‐type‐like bipolar kinetochore–microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1‐dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores.

Recruitment to Golgi membranes of ADP‐ribosylation factor 1 is mediated by the cytoplasmic domain of p23

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi‐resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross‐link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1‐GDP) is shown to interact with p23 whereas ARF1‐GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C‐terminal fragment of ARF1. While a monomeric form of a non‐photolabile p23 peptide does not significantly inhibit formation of the cross‐link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

The Protease-associated Domain and C-terminal Extension Are Required for Zymogen Processing, Sorting within the Secretory Pathway, and Activity of Tomato Subtilase 3 (SlSBT3)

A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn177, Asn203, Asn376, Asn697, and Asn745. SlSBT3 was found to be a very stable enzyme, being fully active at 60 °C and showing highest activity at alkaline conditions with a maximum between pH 7.5 and 8.0. Substrate specificity of SlSBT3 was analyzed in detail, revealing a preference for Gln and Lys in the P1 and P2 positions of oligopeptide substrates, respectively. Similar to bacterial, yeast, and mammalian subtilases, SlSBT3 is synthesized as a preproenzyme, and processing of the prodomain in the endoplasmic reticulum is a prerequisite for passage through the secretory pathway. SlSBT3 S538A and S538C active site mutants accumulated intracellularly as unprocessed zymogens, indicating that prodomain cleavage occurs autocatalytically. The wild-type SlSBT3 protein failed to cleave the prodomain of the S538A mutant in trans, demonstrating that zymogen maturation is an intramolecular process. Distinguishing features of plant as compared with mammalian subtilases include the insertion of a large protease-associated domain between the His and Ser residues of the catalytic triad and the C-terminal extension to the catalytic domain. Both features were found to be required for SlSBT3 activity and, consequently, for prodomain processing and secretion.

The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

hRio1 is an atypical protein kinase of the conserved RIO family. Depletion of hRio1 affects the last step of 18S rRNA maturation and causes defects in recycling of trans-acting factors from pre-40S subunits in the cytoplasm. The kinase activity of hRio1 is essential for recycling of the endonuclease hNob1 and its binding partner hDim2 from pre-40S.

Precursor processing for plant peptide hormone maturation by subtilisin-like serine proteinases

Prohormone processing by subtilases A flower that has gone to seed will drop its petals in a regulated process called abscission. Schardon et al. analyzed the production of the peptide hormone that regulates floral organ abscission in the model plant Arabidopsis thaliana. They used tissue-specific expression of proteinase inhibitors to identify the subtilisin-like proteinases that act as prohormone convertases required for peptide hormone production in plants. Science, this issue p. 1594 Redundant proteases mediate the formation of a peptide signal for the abscission of floral organs in Arabidopsis. Peptide hormones that regulate plant growth and development are derived from larger precursor proteins by proteolytic processing. Our study addressed the role of subtilisin-like proteinases (SBTs) in this process. Using tissue-specific expression of proteinase inhibitors as a tool to overcome functional redundancy, we found that SBT activity was required for the maturation of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION), a peptide signal for the abscission of floral organs in Arabidopsis. We identified three SBTs that process the IDA precursor in vitro, and this processing was shown to be required for the formation of mIDA (the mature and bioactive form of IDA) as the endogenous signaling peptide in vivo. Hence, SBTs act as prohormone convertases in plants, and several functionally redundant SBTs contribute to signal biogenesis.

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p.

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.

Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin

UNLABELLED Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. BIOLOGICAL SIGNIFICANCE The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a proteome wide scale. The high proteome coverage of our proteomics survey allowed us to analyze the regulation of whole protein networks instead of single proteins. We were able to reconstruct the complete degradation pathway of vanillin and to monitor the changes in the energy metabolism of KT2440 induced by vanillin as sole carbon source. Vanillin dehydrogenase (Vdh) was not mandatory for vanillin degradation in KT2440 and may be substituted by other aldehyde dehydrogenases that were up-regulated in a wild-type as well as in a Vdh-deficient strain in the presence of vanillin. Aldehyde dehydrogenases, vanillin specific porins and efflux pump systems identified in study will be interesting targets for optimization of vanillin production in Pseudomonas bacteria. Furthermore, several mechanisms of solvent tolerance were induced by vanillin in KT2440. These include increased abundance of several efflux pump systems, chaperones as well as enzymes involved in cyclopropane fatty acid synthesis and trehalose formation. The present work will deepen the understanding of metabolism of aromatic compounds in P. putida and may lead to a more comprehensive understanding of solvent tolerance mechanisms in Gram-negative bacteria in general. Moreover, it will serve as a basis for further strain developments for a biotechnological production of vanillin in P. putida KT2440 or other Pseudomonas strains, highlighting the role of proteomics surveys as a powerful screening technology.

Light-dependent Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel

The Drosophila phototransduction cascade terminates in the opening of an ion channel, designated transient receptor potential (TRP). TRP has been shown to become phosphorylated in vitro, suggesting regulation of the ion channel through posttranslational modification. However, except for one phosphorylation site, Ser982, which was analyzed by functional in vivo studies (Popescu, D. C., Ham, A. J., and Shieh, B. H. (2006) J. Neurosci. 26, 8570–8577), nothing is known about the role of TRP phosphorylation in vivo. Here, we report the identification of 21 TRP phosphorylation sites by a mass spectrometry approach. 20 phosphorylation sites are located in the C-terminal portion of the channel, and one site is located near the N terminus. All 21 phosphorylation sites were also identified in the inaCP209 mutant, indicating that phosphorylation of TRP at these sites occurred independently from the eye-enriched protein kinase C. Relative quantification of phosphopeptides revealed that at least seven phosphorylation sites were predominantly phosphorylated in the light, whereas one site, Ser936, was predominantly phosphorylated in the dark. We show that TRP phosphorylated at Ser936 was located in the rhabomere. Light-dependent changes in the phosphorylation state of this site occurred within minutes. The dephosphorylation of TRP at Ser936 required activation of the phototransduction cascade.

A model of dirigent proteins derived from structural and functional similarities with allene oxide cyclase and lipocalins

Dirigent proteins impart stereoselectivity on the phenoxy radical‐coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)‐ or (−)‐pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all‐beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N‐linked glycans at the solvent‐exposed protein surface. The model shows AtDIR6 as an eight‐stranded antiparallel β‐barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand‐binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product.