Bradley J.S.C. Olson

Assays for Determination of Protein Concentration

Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high‐throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS‐PAGE sample buffer that is used for equal loading of SDS‐PAGE gels, which is reliable, inexpensive, and quick.

Evolution of an Expanded Sex-Determining Locus in Volvox

Revealing Volvox Female and male gametes of the green alga, Volvox, significantly differ in size. Those of Chlamydomonas, another green algae from a lineage that separated from Volvox some 200 million years ago, are the same size. We know sex in Chlamydomonas is governed by a sex-determining locus called MT. In a detailed comparison of the MT loci of Volvox and Chlamydomonas, Ferris et al. (p. 351) found that although MT has retained some similarity in gene order, its composition has greatly changed between the two species. In Volvox, new genes have been coopted into this locus and show sex-specific expression. Mating loci among green algae show conserved gene order, but also have many unique features that may explain gamete size differences. Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non–sex-related genes.

Plastid division: across time and space

Plastid division is executed by the coordinated action of at least two molecular machineries--an internal machinery situated on the stromal side of the inner envelope membrane that was contributed by the cyanobacterial endosymbiont from which plastids evolved, and an external machinery situated on the cytosolic side of the outer envelope membrane that was contributed by the host. Here we review progress in defining the components of the plastid division complex and understanding the mechanisms of envelope constriction and division-site placement in plants. We also highlight recent work identifying the first molecular linkage between the internal and external division machineries, shedding light on how their mid-plastid positioning is coordinated across the envelope membranes. Little is known about the mechanisms that regulate plastid division in plant cells, but recent studies have begun to hint at potential mechanisms.

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZ genes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type (WT). Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2-1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZ triple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex.

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

GTP-dependent Heteropolymer Formation and Bundling of Chloroplast FtsZ1 and FtsZ2

Bacteria and chloroplasts require the ring-forming cytoskeletal protein FtsZ for division. Although bacteria accomplish division with a single FtsZ, plant chloroplasts require two FtsZ types for division, FtsZ1 and FtsZ2. These proteins colocalize to a mid-plastid Z ring, but their biochemical relationship is poorly understood. We investigated the in vitro behavior of recombinant FtsZ1 and FtsZ2 separately and together. Both proteins bind and hydrolyze GTP, although GTPase activities are low compared with the activity of Escherichia coli FtsZ. Each protein undergoes GTP-dependent assembly into thin protofilaments in the presence of calcium as a stabilizing agent, similar to bacterial FtsZ. In contrast, when mixed without calcium, FtsZ1 and FtsZ2 exhibit slightly elevated GTPase activity and coassembly into extensively bundled protofilaments. Coassembly is enhanced by FtsZ1, suggesting that it promotes lateral interactions between protofilaments. Experiments with GTPase-deficient mutants reveal that FtsZ1 and FtsZ2 form heteropolymers. Maximum coassembly occurs in reactions containing equimolar FtsZ1 and FtsZ2, but significant coassembly occurs at other stoichiometries. The FtsZ1:FtsZ2 ratio in coassembled structures mirrors their input ratio, suggesting plasticity in protofilament and/or bundle composition. This behavior contrasts with that of α- and β-tubulin and the bacterial tubulin-like proteins BtubA and BtubB, which coassemble in a strict 1:1 stoichiometry. Our findings raise the possibility that plasticity in FtsZ filament composition and heteropolymerization-induced bundling could have been a driving force for the coevolution of FtsZ1 and FtsZ2 in the green lineage, perhaps arising from an enhanced capacity for the regulation of Z ring composition and activity in vivo.

Regulation of the Chlamydomonas Cell Cycle by a Stable, Chromatin-Associated Retinoblastoma Tumor Suppressor Complex

The retinoblastoma (RB) pathway is a conserved eukaryotic cell cycle regulator that is thought to control cell cycle progression through periodic dissociation of the repressor protein, RB, from the activator proteins E2F and DP. This study shows that in the unicellular alga Chlamydomonas, the cell cycle is regulated by a constitutively chromatin-bound RB-E2F-DP ternary complex whose subunits do not undergo periodic dissociation. We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit—RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle–regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.

The Arabidopsis translocator protein (AtTSPO) is regulated at multiple levels in response to salt stress and perturbations in tetrapyrrole metabolism

BackgroundThe translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. Arabidopsis thaliana contains a single TSPO/PBR-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized.ResultsTo test if the TSPO N-terminal extension targets it to organelles, we fused three potential translational start sites in the TSPO cDNA to the N-terminus of GFP (AtTSPO:eGFP). The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown. Full-length AtTSPO:eGFP fusion protein was found in the endoplasmic reticulum and in vesicles of unknown identity when plants were grown in standard conditions. However, full length AtTSPO:eGFP localized to chloroplasts when grown in the presence of 150 mM NaCl, conditions of salt stress. In contrast, when AtTSPO:eGFP was truncated to the second or third start codon at amino acid position 21 or 42, the fusion protein co-localized with a mitochondrial marker in standard conditions. Using promoter GUS fusions, qRT-PCR, fluorescent protein tagging, and chloroplast fractionation approaches, we demonstrate that AtTSPO levels are regulated at the transcriptional, post-transcriptional and post-translational levels in response to abiotic stress conditions. Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed. Mutations in tetrapyrrole biosynthesis genes and the application of chlorophyll or carotenoid biosynthesis inhibitors also affect AtTSPO expression.ConclusionOur data suggest that AtTSPO plays a role in the response of Arabidopsis to high salt stress. Salt stress leads to re-localization of the AtTSPO from the ER to chloroplasts through its N-terminal extension. In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants. Thus, we propose that AtTSPO may play a role in transporting tetrapyrrole intermediates during salt stress and other conditions in which tetrapyrrole metabolism is compromised.

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. DOI: http://dx.doi.org/10.7554/eLife.10767.001