Identification of gene encoding Plasmodium knowlesi phosphatidylserine decarboxylase by genetic complementation in yeast and characterization of in vitro maturation of encoded enzyme

The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ～5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.     

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.     

An early event in the transport mechanism of LacY protein: interaction between helices V and I

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg¹⁴⁴ and Trp¹⁵¹, two residues that play direct roles in sugar recognition and binding, as well as Cys¹⁵⁴, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro²⁸ → Cys/Cys¹⁵⁴, Pro²⁸ → Cys/Val¹⁵⁸ → Cys, and Phe²⁹ → Cys/Val¹⁵⁸ → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)₂-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)₂-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.     

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria

As a consequence of their bacterial origin, mitochondria contain β-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the β-barrel assembly machinery (BAM) required for biogenesis of β-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial β-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only β-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial β-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of β-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of β-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.