Cellular expression and alternative splicing of SLC25A23, a member of the mitochondrial Ca2+-dependent solute carrier gene family

Phospholipase D (PLD) is a ubiquitous enzyme in eukaryotes that participates in various cellular processes. Its catalytic domain is characterized by two HKD motifs in the C-terminal part. Until now, two subfamilies were recognized based on their N-terminal domain structure. The first has a PX domain in combination with a PH domain and is designated as PXPH-PLD. Members of the second subfamily, named C2-PLD, have a C2 domain and have, so far, only been found in plants. Here we describe a novel PLD subfamily that we identified in Phytophthora, a genus belonging to the class oomycetes and comprising many important plant pathogens. We cloned Pipld1 from Phytophthora infestans and retrieved full-length sequences of its homologues from Phytophthora sojae and Phytophthora ramorum genome databases. Their promoters contain two putative regulatory elements, one of which is highly conserved in all three genes. The three Phytophthora pld1 genes encode nearly identical proteins of around 1807 amino acids, with the two characteristic HKD motifs in the C-terminal part. Homology of the predicted proteins with known PLDs however is restricted to the two catalytic HKD motifs and adjacent domains. In the N-terminal part Phytophthora PLD1 has a PX-like domain, but it lacks a PH domain. Instead the N-terminal region contains five putative membrane spanning domains suggesting that Phytophthora PLD1 is a transmembrane protein. Since Phytophthora PLD1 cannot be categorized in one of the two existing subfamilies we propose to create a novel subfamily named PXTM-PLD.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Pigment-pigment interactions in Lhca4 antenna complex of higher plants photosystem I

The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 A) versus A4 and A5 (12.2 A) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.     

Slowly reversible de-epoxidation of lutein-epoxide in deep shade leaves of a tropical tree legume may 'lock-in' lutein-based photoprotection during acclimation to strong light

The kinetics of response to strong light have been examined in deeply shaded leaves of the tropical tree legume (Inga sp.) which have extraordinarily high levels of the alpha-xanthophyll lutein-epoxide that are co-located in pigment-protein complexes of the photosynthetic apparatus with the beta-xanthophyll violaxanthin. As in other species, rapidly reversible photoprotection (measured as non-photochemical chlorophyll fluorescence quenching) is initiated within the time frame of sun-flecks (minutes), before detectable conversion of violaxanthin to antheraxanthin or zeaxanthin. Photoprotection is stabilized within hours of exposure to strong light by simultaneously engaging the reversible violaxanthin cycle and a slowly reversible conversion of lutein-epoxide to lutein. It is proposed that this lutein 'locks in' a primary mechanism of photoprotection during photoacclimation in this species, converting efficient light-harvesting antennae of the shade plant into potential excitation dissipating centres. It is hypothesized that lutein occupies sites L2 and V1 in light-harvesting chlorophyll protein complexes of photosystem II, facilitating enhanced photoprotection through the superior singlet and/or triplet chlorophyll quenching capacity of lutein.     

Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization

Mucolipin-1 is a 65-kDa membrane protein encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We studied the subcellular localization of wild-type and three different mutant forms (T232P, F408del and F465L) of mucolipin by expressing Myc-tagged proteins in HeLa cells. The overexpressed wild-type mucolipin colocalizes to late endocytic structures and induces an aberrant distribution of these compartments. F408del and F465L MLIV mutant proteins show a distribution similar to the wild-type protein, whereas T232P is retained in the endoplasmic reticulum. Among the mutants, only F408del induces a redistribution of the late endocytic compartment. These findings suggest that the overexpression of the mucolipin cation channel influences the dynamic equilibrium of late endocytic compartments.     

Stark effect measurements on monomers and trimers of reconstituted light-harvesting complex II of plants

The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states-monomers and trimers-with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most approximately 25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.     

Photosynthesis research in Italy: a review

This historical review was compiled and edited by Giorgio Forti, whereas the other authors of the different sections are listed alphabetically after his name, below the title of the paper; they are also listed in the individual sections. This review deals with the research on photosynthesis performed in several Italian laboratories during the last 50 years; it includes research done, in collaboration, at several international laboratories, particularly USA, UK, Switzerland, Hungary, Germany, France, Finland, Denmark, and Austria. Wherever pertinent, references are provided, especially to other historical papers in Govindjee et al. [Govindjee, Beatty JT, Gest H, Allen JF (eds) (2005) Discoveries in Photosynthesis. Springer, Dordrecht]. This paper covers the physical and chemical events starting with the absorption of a quantum of light by a pigment molecule to the conversion of the radiation energy into the stable chemical forms of the reducing power and of ATP. It describes the work done on the structure, function and regulation of the photosynthetic apparatus in higher plants, unicellular algae and␣in photosynthetic bacteria. Phenomena such as photoinhibition and the protection from it are also included. Research in biophysics of photosynthesis in Padova (Italy) is discussed by G.M. Giacometti and G.␣Giacometti (2006).