Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin  subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin ␣ subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin  subunit results in the complete absence of the ␣ subunit and the linkers. Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain. Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit. Phycocyanin assembly begins with the formation of the ␣ heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods. The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo.Light harvesting in cyanobacteria is mediated by the phycobilisomes, which are complex protein structures located on the surface of the photosynthetic membrane (20). The major structural components of the phycobilisome are the biliproteins, which contain covalently attached bilin chromophores that constitute a resonance energy transfer pathway. Light energy in the 500-to 650-nm range can be absorbed by different classes of biliproteins and is rapidly and efficiently transferred through the phycobilisome to chlorophyll complexes in the photosynthetic membrane. The three major classes of biliproteins are distinguished by their spectral properties. The allophycocyanins (AP [ max ϭ 650 to 665 nm]) are located in the core of the phycobilisome, which is in direct contact with chlorophyll complexes in the membrane. Phycocyanin (PC [ max ϭ 617 nm]) is found in the rod substructures that are attached to the phycobilisome cores. A third major biliprotein, phycoerythrin (PE [ max ϭ 565 nm]), is synthesized in some cyanobacteria and is attached to PC at the periphery of the rod substructures. Each biliprotein has the same subunit organization that is based on a heterodimer (called a monomer by convention) composed of ␣ and  subunits (11,16,(37)(38)(39). Monomers are assembled into disc-like trimers with a central channel, which then stack to form a hexamer in the PC and PE biliproteins. Hexamers are associated with single copies of linker proteins that direct their assembly into the rod substructures. The organization of biliproteins within the phycobilisome structure establishes an energy transfer pathway, PE to PC to AP to chlorophyll, that operates at close to 100% effici...
Tuberous sclerosis complex (TSC) is a common genetic disorder in which affected individuals can develop mental retardation, developmental brain defects, and seizures. Two genetic loci are responsible for TSC: TSC1 on chromosome 9q and TSC2 on chromosome 16p. Here, we report our analysis of TSC1 (hamartin) and TSC2 (tuberin) protein expression in the central nervous system (CNS). Both tuberin and hamartin are expressed in neurons and astrocytes where they physically interact. In the mouse cerebellum in vivo, tuberin predominantly localizes to the perinuclear region of the Purkinje cell, whereas hamartin is distributed along neuronal or astrocytic processes. In contrast, both hamartin and tuberin demonstrate similar neuronal expression patterns in pure neuronal cultures in vitro. Additionally, hamartin is highly expressed in astrocytes in mixed neuron-glia cultures in vitro, suggesting that hamartin may be important for astrocyte growth control. Unlike tuberin, loss of hamartin expression was not observed in sporadic astrocytomas. These results suggest that tuberin and hamartin may differentially contribute to the CNS pathology in TSC.
Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membraneassociated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin  subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin ␣ and  subunits assemble with endogenous phycobilisome components. The phycocyanin ␣ subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.Phycobilisomes from the unicellular cyanobacterium Synechocystis sp. strain 6803 consist of the AP and PC biliproteins. We have recently characterized a PC-minus mutant, Synechocystis sp. strain 6803 (strain 4R) (15). This strain synthesizes phycobilisome cores without rods because of a single-base insertion in the cpcB gene, producing a truncated PC  subunit that leads to a complete absence of both PC subunits. The PC-minus phenotype of 4R suggested that this transformable cyanobacterium (20) would be a suitable genetic host for the introduction of heterologous biliprotein genes in studies focused on early events of biliprotein biosynthesis. We are particularly interested in analyzing structural differences between PC and PE subunits that direct recognition-dependent processes such as subunit assembly and chromophore attachment. These questions can be addressed by site-directed mutagenesis and protein domain exchange in a heterologous transformation system that employs the PC-minus strain of Synechocystis sp. strain 6803 as a host for expression of the cpc and cpe genes from Synechocystis sp. strain 6701 (1, 2, 10, 12). This report is an analysis of Synechocystis sp. strain 6803 transformants that express the cpcBA operon from Synechocystis sp. strain 6701. The results validate the heterologous transformation system as a research tool and demonstrate the rescue of the endogenous PC ␣ subunit in 4R by heterologous assembly. MATERIALS AND METHODSBacterial strains and culture media. Synechocystis sp. strain 6803 (wild-type [WT] and 4R strains) were grown as previously described (15). Liquid and solid media were supplemented with glucose (20 mM)...
SummaryLight-har vesting in cyanobacteria and red algae is a function of the biliproteins, which have covalently bound bilin chromophores. The biliproteins are assembled with linker proteins into the phycobilisome, a large complex that resides on the surface of the photosynthetic membranes. Early steps in the phycobilisome assembly pathway include the folding of biliprotein ␣-and -subunits, covalent modification of subunits by bilin attachment and formation of the primary assembly unit, the ␣ heterodimer. The potential role of bilins in subunit structure and assembly is examined in this study by site mutagenesis of biliprotein genes. Phycocyanin subunits from Synechocystis sp. 6701 that were unable to bind chromophores at specific sites were generated by changing the codons for bilin-binding cysteines to alanine residues. The altered genes were then expressed in a phycocyaninminus mutant of the transformable Synechocystis sp. strain 6803. Single and multiple chromophore deletions cause specific and reproducible variations in phycobilisome-associated phycocyanin that do not correlate with transcript levels. Sedimentation equilibrium studies with purified proteins showed that bilin absence reduces the strength of ␣ interaction in the heterodimer. These results suggest that phycocyanin instability in bilin-deletion mutants is a consequence of diversion of unassembled ␣-and -subunits to a degradation pathway. Attachment of the central bilin, which is common to all biliprotein subunits, may facilitate ␣ interaction by completing the final stage of subunit folding and stabilizing the contact domains of binding partners in the heterodimer.
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