The carbon concentrating mechanism in Chlamydomonas reinhardtii: Finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3-, the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed. © 2014 Springer Science+Business Media Dordrecht

Influenza matrix protein M1

Die Aufklärung der Prozesse, die zur Zusammensetzung des Influenza A Virus führen, ist Bestandteil für die Bekämpfung dieser Infektionskrankheit. Der Viruspartikel setzt sich aus einer Hülle, der darunter liegenden Matrix und dem Genom zusammen. Das Genom ist als Bündel aus acht Ribunucleoproteinkomplexen organisiert. Die Hülle besteht aus einer Membran, die mit Sphingomyelin und Cholesterol angereichert ist und den darin eingebetteten Membranproteinen Hämagglutinin, Neuraminidase und dem Protonenkanal M2. Die unter der Hülle liegende Matrix wird von einem einzigen Influenzaprotein formiert: Dem Matrixprotein M1. Es spielt eine Schlüsselrolle im Replikationszyklus des Virus in der Zelle. Es interagiert mit dem genetischen Material, mit den Membranproteinen und der Lipidmembran der Hülle. Die vorliegende Arbeit gibt Auskunft, welche Lipide eine Rolle in der M1-MembranWechselwirkung spielen. Die Liste der identifizierten Lipide umfasst neben dem bereits bekannten Phosphatidylserin auch Phosphatidylglycerol und Phosphatidsäure. Verschiedene Phosphatidylinositole konnten ebenfalls identifiziert werden. Als stärkster M1 Bindungspartner trat dabei Phosphatidylinositol-4-Phosphat zutage. Weitere auf Mutanten basierende Untersuchungen zeigten, dass der membranbindende Bereich nicht auf eine einzelne Domäne in M1 festgelegt werden kann. Die N-terminale M1-Domäne mit ihrem Oberflächen-exponierten, positiv geladenen Areal und die C-terminale Domäne interagierten mit Modellmembranen. Das Resultat dieser Interaktionen konnte mittels mikroskopischer Untersuchungen an gigantischen unilamellaren Vesikeln dokumentiert werden. Für M1 und für eine Mutante, die nur aus der N-terminalen M1-Domäne besteht, konnte eine von anderen viralen Proteinen unabhängige homooligomere Organisation auf der Membran gezeigt werden. Diese M1-Cluster könnten während der Zusammensetzung des Viruspartikels als Fundament für die Eingliederung aller weiteren viralen Komponenten dienen.about the assembly process of the influenza A virus particle is essential for the development of effective approaches for prevention and treatment of this virus infection. The virus particle consists of an envelope, an underlying matrix, and the encapsulated genome. The genetic material is organized as bundle of eight ribonucleoprotein complexes that encode for eleven proteins. The envelope consists of a lipid bilayer that is enriched in sphingomyelin and cholesterol. The viral spike proteins, hemagglutinin and neuraminidase, as well as the proton channel M2 are embedded into this membrane. The matrix can be found below the envelope. It is formed by one single protein, the matrix protein M1. M1 plays a crucial role during the replication of the virus in the cell. It interacts with the genetic material, with the envelope proteins and with the lipid bilayer of the envelope. The results of this study reveal in detail which lipids are targeted by M1. The set of identified lipids contains phosphatylglycerol and phosphatidic acids as new binding partners, beside the known phophatidylserine. Additionally, several phosphatidylinositols were identified. Phosphatidylinositol-4-phosphate was the strongest binding partner from this group. Mutant-based analysis revealed that M1 owns more than one membrane binding site. The positively charged area in the N-terminal and the C-terminal domain mediated membrane association of the respective mutant protein. The final constitution of M1 on the membrane was characterized by confocal fluorescence microscopy on giant unilamellar vesicles. Full length M1 and a mutant that consisted only of the N-terminal part of M1 showed lateral clustering of homooligomers on the vesicle surface. The clusters formed independently of any other viral component. A function as fundament for the incorporation of the other viral components can be assumed for these clusters

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions

The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3-, the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed. © 2014 Springer Science+Business Media Dordrecht