Birgitta Norling

Ubiquinone biosynthesis by the microsomal fraction from rat liver

The distribution and biosynthesis of ubiquinone were investigated in vivo in rats and using liver slices. In addition to mitochondria, Golgi vesicles and lysosomes also contain large amounts of this lipid, and even the plasma membrane, peroxisomes and microsomes demonstrate easily measurable amounts. The spectral and chromatographic properties of microsomal ubiquinone were identical to those of its mitochondrial counterpart. When pentane was used to deplete beef heart submitochondrial particles of ubiquinone, NADH and succinate oxidase activities could be restored by reincorporation of microsomal ubiquinone. Injection of [3H]mevalonate into the portal vein of rats and incubation of liver slices with [3H]mevalonate and [3H]- and [14C]tyrosine demonstrated that labeling of mitochondrial ubiquinone was initially much lower than labeling of the microsomal lipid. Furthermore, intraportal injection of [3H]mevalonate resulted in the rapid appearance of labeled ubiquinone in the blood. These results indicate that ubiquinone is synthesized not only in mitochondria, but also on the endoplasmic reticulum of rat liver.

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.

Proteomics of Synechocystis sp. Strain PCC 6803 Identification of Plasma Membrane Proteins

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F0 membrane part of the ATP synthase.

2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803

Aqueous polymer two‐phase partitioning in combination with sucrose density centrifugation offered, for the first time, a 2D‐separation method for the isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis 6803 without any cross‐contaminations. The purity of the membrane fractions was verified by immunoblot analysis using antibodies against membrane‐specific marker proteins. As an initiation of a proteomics project, two prominent proteins, which were observed only in the plasma membrane (Slr1513, a hypothetical protein, and HofG, a general secretion pathway protein), or in the thylakoid membrane (PsaE, a photosystem I protein, and NdhH, a subunit of NADH dehydrogenase), were identified.

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (α‐tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP‐Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ‐10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQHZ efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.

Isolation of Outer Membrane of Synechocystis sp. PCC 6803 and Its Proteomic Characterization

In this report, we describe a newly developed method for isolating outer membranes from Synechocystis sp. PCC 6803 cells. The purity of the outer membrane fraction was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. We investigated the protein composition of the outer membrane using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by database identification. Forty-nine proteins were identified corresponding to 29 different gene products. All of the identified proteins have a putative N-terminal signal peptide. About 40% of the proteins identified represent hypothetical proteins with unknown function. Among the proteins identified are a Toc75 homologue, a protein that was initially found in the outer envelope of chloroplasts in pea, as well as TolC, putative porins, and a pilus protein. Other proteins identified include ABC transporters and GumB, which has a suggested function in carbohydrate export. A number of proteases such as HtrA were also found in the outer membrane of Synechocystis sp. PCC 6803.

Proteomic analysis of plasma membranes of cyanobacterium Synechocystis sp. Strain PCC 6803 in response to high pH stress.

Cyanobacteria are unique prokaryotes possessing plasma-, outer- and thylakoid membranes. The plasma membrane of a cyanobacterial cell serves as a crucial barrier against its environment and is essential for biogenesis of cyanobacterial photosystems. Previously, we have identified 79 different proteins in the plasma membrane of Synechocystis sp. Strain PCC 6803 based on 2D- and 1D- gels and MALDI-TOF MS. In this work, we have performed a proteomic study screening for high-pH-stress proteins in Synechocystis. 2-D gel profiles of plasma membranes isolated from both control and high pH-treated cells were constructed and compared quantitatively based on different protein staining methods including DIGE analysis. A total of 55 differentially expressed protein spots were identified using MALDI-TOF MS and MALDI-TOF/TOF MS, corresponding to 39 gene products. Twenty-five proteins were enhanced/induced and 14 reduced by high pH. One-third of the enhanced/induced proteins were transport and binding proteins of ABC transporters including 3 phosphate transport proteins. Other proteins include MinD involved in cell division, Cya2 in signaling and proteins involved in photosynthesis and respiration. Furthermore, among these proteins regulated by high pH, eight were found to be hypothetical proteins. Functional significance of the high-pH-stress proteins is discussed integrating current knowledge on cyanobacterial cell physiology.

Proteomics of Synechocystis sp. PCC 6803

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI‐TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.

Lack of ability of trypsin-treated mitochondrial F1-ATPase to bind the oligomycin-sensitivity conferring protein (OSCP).

Soluble beef‐heart mitochondrial F1‐ATPase modified in its α‐subunit by mild trypsin treatment (α′‐F1) can no longer bind oligomycin‐sensitivity conferring protein (OSCP) but is still capable of binding to F1‐depleted submitochondrial particles, giving rise to a maximally oligomycin‐sensitive ATPase, provided the particles contain their native complement of OSCP. When OSCP is removed from the particles, α′‐F1 can still bind to the particles, but added OSCP induces only a low degree of oligomycin sensitivity. The possible role of OSCP in the functional coupling of the catalytic (F1) and H+‐translocating (Fo) moieties of mitochondrial ATPase is discussed. The results suggest a functional similarity between the OSCP component of mitochondrial ATPase and the δ‐subunit of E. coli ATPase, which is in accordance with the structural homology recently found to exist between the two polypeptides.

Proteomics of Synechocystis sp. PCC 6803. Identification of novel integral plasma membrane proteins.

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI‐TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.