Search

The segregating unit of mtDNA is a protein–DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected ≈20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Δ mutation increases the petite-inducing phenotype of an abf2Δ mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of ρ + mtDNA.

Studies demonstrating differences in chloroplast structure and biochemistry have been used to formulate hypotheses concerning the origin of algal plastids. Genetic and biochemical experiments indicate that significant variation occurs in ribulose-1,5-bisphosphate carboxylase (Rubisco) when supertaxa of eukaryotic algae are compared. These differences include variations in the organelle location of the genes and their arrangement, mechanism of Rubisco synthesis, polypeptide immunological reactivity and sequence, as well as efficacy of substrate (ribulose bisphosphate and CO2) binding and inhibitor (6-phosphogluconate) action. The structure-function relationships observed among chromophytic, rhodophytic, chlorophytic and prokaryotic Rubisco demonstrate that: (a) similarities among chromophytic and rhodophytic Rubisco exist in substrate/inhibitor binding and polypeptide sequence, (b) characteristic differences in enzyme kinetics and subunit polypeptide structure occur among chlorophytes, prokaryotes and chromophytes/rhodophytes, and (c) there is structural variability among chlorophytic plant small subunit polypeptides, in contrast to the conservation of this polypeptide in chromophytes and rhodophytes. Taxa-specific differences among algal Rubisco enzymes most likely reflect the evolutionary history of the plastid, the functional requirements of each polypeptide, and the consequences of encoding the large and small subunit genes in the same or different organelles.

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5′ non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg(2+) have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO(3) and Mg(2+) for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO(2) and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO(2) fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.

Ribulose-1,5-bisphosphate carboxylase (Rubisco) catalyzes the initial CO2-fixing reaction of photosynthesis in prokaryotes and eukaryotes. The active holoenzyme consists of 8 large subunits (LS) and 8 small subunits (SS), except in R. rubrum which contains only two large subunits (1). In chlorophytic plants (chlorophyll a,b), rbcL (large subunit gene) is chloroplast DNA encoded where as the small subunit genes (rbcS) are nuclear encoded and the small subunit gene products are post-translationally transported into the plastid. This organization of rbcL and rbcS is not representative of all plants. In the chromophytic (chl.a,c) alga O1isthodiscus luteus, both subunits are coded for by chloroplast DNA (ctDNA) (2). The proximity and arrangement (5′-LS-SS-3′) of these genes are identical to that seen in blue-green algae and in Cyanophora Chapaudoxa. Since O. luteus is a eukaryotic alga with a prokaryotic organization of rbcL and rbcS, the enzymology and mechanism of biosynthesis of the O. luteus Rubisco are expected to be unique when compared to the enzyme from other photoautotrophs. In this study, the biochemical analysis of Rubisco has been extended to include the enzyme from the rhodophytic (red) alga Griffithsia pacifica, in addition to using antibodies and proteases to determine the structural relatedness of this enzyme and its subunits among chromophytic, rhodophytic and chlorophytic plants.

Ribulose-1,5-bisphosphate carboxylase (Rubisco) from the algae Olisthodiscus luteus (chromophyte) and Griffithsia pacifica (rhodophyte) are remarkably similar to each other. However, both enzymes differ significantly in the structure and function when compared to Rubisco from green algae and land plants. Analysis of purified Rubisco from O. luteus and G. pacifica indicates that the size of the holoenzyme and stoichiometry of the 55 and 15 kilodalton subunit polypeptides are approximately 550 kilodaltons and eight:eight for both algae. Antigenic determinants are highly conserved between the O. luteus and G. pacifica enzymes and differ from those of the spinach subunit polypeptides. Sequence similarity between the two algal large subunits has been further confirmed by one-dimensional peptide mapping. Substrate ribulose bisphosphate has no effect on the rate of CO(2)/Mg(2+) activation of O. luteus and G. pacifica enzymes which contrasts to the extensive inhibition of spinach Rubisco activation at similar concentrations of this compound. In addition, the Michaelis constant for CO(2) and the inhibition constant for 6-phosphogluconate are similar for the O. luteus and G. pacifica catalyzed carboxylation reaction. Both values are intermediate to those observed for the tight binding spinach enzyme and weak binding prokaryotic (Rhodospirillum rubrum) enzyme. The biochemical similarities documented between O. luteus and G. pacifica may be due to a common evolutionary origin on the chromophytic and rhodophytic chloroplast but could also result from the fact that both subunit polypeptides are chloroplast DNA encoded in these algal taxa.