Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides wild-type cells. PufX is associated with the reaction center-light harvesting 1 (RC-LH1) core complex and plays a key role in lateral ubiquinone/ubiquinol transfer. We have determined the PufX/RC stoichiometry by quantitative Western blot analysis and RC photobleaching. Independent of copy number effects and growth conditions, one PufX molecule per RC was observed in native membranes as well as in detergent-solubilized RC-LH1 complexes which had been purified over sucrose gradients. Surprisingly, two gradient bands with significantly different sedimentation coefficients were found to have a similar subunit composition, as judged by absorption spectroscopy and protein gel electrophoresis. Gel filtration chromatography and electron microscopy revealed that these membrane complexes represent a monomeric and a dimeric form of the RC-LH1 complex. Since PufX is strictly required for the isolation of dimeric core complexes, we suggest that PufX has a central structural role in forming dimeric RC-LH1 complexes, thus allowing efficient ubiquinone/ubiquinol exchange through the LH1 ring surrounding the RC.     

Dimerisation of the Rhodobacter sphaeroides RC-LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide

In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC-LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC-LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC-LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC-LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC-LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC-LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.     

Experimental evidence that the membrane-spanning helix of PufX adopts a bent conformation that facilitates dimerisation of the Rhodobacter sphaeroides RC-LH1 complex through N-terminal interactions

The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC-LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC-LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC-LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and β polypeptides on the other half of the dimer.     

Protein-Induced Membrane Curvature Investigated through Molecular Dynamics Flexible Fitting

In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)₂ protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of ∼250-550 Å, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even though the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Preferential Incorporation of Coloured-carotenoids Occurs in the LH2 Complexes From Non-sulphur Purple Bacteria Under Carotenoid-limiting Conditions

The effect of growing Rhodopseudomonas (Rps.) acidophila and Rps. palustris in the presence of different concentrations of the carotenoid (Car) biosynthetic inhibitor diphenylamine (DPA) has been investigated. Growth with sub-maximal concentrations of DPA induces Car limitation. The exact response to DPA is species dependent. However, both Rps. acidophila and Rps. palustris respond by preferentially incorporating the limiting amount of coloured Cars into their LH2 complexes at the expense of the RC-LH1 complexes. As inhibition by DPA becomes more severe there is an increase in the percentage of Cars with reduced numbers of conjugated C=C bonds. The effect of this changed Car composition on the structure and function of the antenna complexes has been investigated using absorption, fluorescence, CD and Raman spectroscopies. The results show that although the presence of Car molecules is important for the stability of the LH2 complexes that the overall native structure can be maintained by the presence of many different Cars.     

The solution structure of the PufX polypeptide from Rhodobacter sphaeroides

PufX organises the photosynthetic reaction centre–light harvesting complex 1 (RC–LH1) core complex of Rhodobacter sphaeroides and facilitates quinol/quinone exchange between the RC and cytochrome bc₁ complexes. The structure of PufX in organic solvent reveals two hydrophobic helices flanked by unstructured termini and connected by a helical bend. The proposed location of basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal, with the GXXXG motifs in positions unsuitable as direct drivers of dimerisation of the RC–LH1 complex. The N-terminal helix is predicted to extend 40 Å along the membrane interface.     

Structure, function and interactions of the PufX protein

The PufX protein is an important component of the reaction centre-light-harvesting 1 (RC-LH1) complex of Rhodobacter species of purple photosynthetic bacteria. Early studies showed that removal of the PufX protein causes changes in the structure of the RC-LH1 complex that result in a loss of the capacity for photosynthetic growth, and that this loss can be overcome though further mutations that change the structure of the LH1 antenna. More recent studies have examined interactions of the PufX protein with other components of the RC-LH1 complex. This review considers our current understanding of the structure and function of the PufX protein, how this protein interacts with other components of the photosynthetic membrane, and its influence on the oligomeric state of the RC-LH1 complex and the larger-scale architecture of the photosynthetic membrane.     

Characterization of a Highly Purified, Fully Active, Crystallizable RC–LH1–PufX Core Complex from Rhodobacter sphaeroides

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1–PufX complex (RC–LH1–PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQ_A → D⁺Q_A^-) in the RC as a function of light intensity. The RC–LH1–PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.     

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6 Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.     

Construction and analysis of a human testis/sperm-enriched interaction network: Unraveling the PPP1CC2 interactome

BackgroundPhosphoprotein phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a PPP1CC tissue-specific alternative splice restricted to testicular germ cells and spermatozoa, is essential for spermatogenesis and spermatozoa motility. The key to understand PPP1CC2 regulation lies on the characterization of its interacting partners.MethodsWe construct a testis/sperm-enriched protein interaction network and analyzed the topological properties and biological context of the network. Further the interaction of a potential target for pharmacological intervention was validated in human spermatozoa.ResultsA total of 1778 proteins and 32,187 interactions between them were identified in the testis/sperm-enriched network. The network analysis revealed the members of functional modules that interact more tightly with each other. In the network, PPP1CC was located in the fourth maximum core part (k = 41) and had 106 direct interactors. Sixteen PPP1CC interactors were involved in spermatogenesis-related categories. Also, PPP1CC had 50 direct interactors, highly interconnected and many of them part of the network maximum core (k = 44), associated with motility-related annotations, including several previously uncharacterized interactors, such as, LMNA, JAK2 and RIPK3.ConclusionsIn this study we integrated tissue-specific protein expression and protein-protein interaction data in order to identify key PPP1CC2 complexes for male reproductive functions.One of the most intriguing interactors was A-kinase anchor protein 4 (AKAP4), a testis-specific protein related to infertility phenotypes and involved in all major motility-related annotations.General significanceWe demonstrated for the first time the interaction between PPP1CC2 and AKAP4 in human spermatozoa and the potential of the complex as contraceptive target.     

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.     

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

An efficient synthesis and biological screening of benzofuran and benzo[d]isothiazole derivatives for Mycobacterium tuberculosis DNA GyrB inhibition

A series of twenty eight molecules of ethyl 5-(piperazin-1-yl)benzofuran-2-carboxylate and 3-(piperazin-1-yl)benzo[d]isothiazole were designed by molecular hybridization of thiazole aminopiperidine core and carbamide side chain in eight steps and were screened for their in vitro Mycobacterium smegmatis (MS) GyrB ATPase assay, Mycobacterium tuberculosis (MTB) DNA gyrase super coiling assay, antitubercular activity, cytotoxicity and protein–inhibitor interaction assay through differential scanning fluorimetry. Also the orientation and the ligand–protein interactions of the top hit molecules with MS DNA gyrase B subunit active site were investigated applying extra precision mode (XP) of Glide. Among the compounds studied, 4-(benzo[d]isothiazol-3-yl)-N-(4-chlorophenyl)piperazine-1-carboxamide (26) was found to be the most promising inhibitor with an MS GyrB IC₅₀ of 1.77 ± 0.23 μM, 0.42 ± 0.23 against MTB DNA gyrase, MTB MIC of 3.64 μM, and was not cytotoxic in eukaryotic cells at 100 μM. Moreover the interaction of protein–ligand complex was stable and showed a positive shift of 3.5 °C in differential scanning fluorimetric evaluations.     

The effects of age,energy and protein intake on protein turnover and the expression of proteolysis-related genes in the broiler breeder hen

A study was conducted to determine the changes that occur to proteolysis and related genes due to age, protein, and energy intake in high-yield broiler breeder hens (Gallus gallus). Cobb 700 broiler breeders were randomly assigned to one of six diets in a 2 × 3 factorial fashion. Two levels of energy (390 and 450 kcal/day) and three levels of protein (22, 24, and 26 g CP/day) were utilized. Protein turnover was determined in the left pectoralis at 22, 26, 31 and 44 weeks. Relative mRNA expression of calpain 2 (CAPN2), proteasome C2 subunit (PSMA1), and F box protein 32 (FBXO32) were determined via RT-PCR at 20, 25, and 44 weeks. Contrasts indicate fractional synthesis rate (FSR) and FBXO32 increase to a maximum at 25–26 weeks and a decrease thereafter. A significant drop in PSMA1 and FBXO32 was observed between 25 and 44 weeks and matched the decrease observed in FBR. No differences were detected in the levels of fractional synthesis and degradation, or the expression of CAPN2, PSMA1, and FBXO32, due to protein or energy intake. In summary, protein turnover was upregulated during the transition into sexual maturity and decreased thereafter. The observed changes in degradation appeared to be mediated by the ubiquitin–proteasome pathway.     

Structure of the dimeric RC–LH1–PufX complex from Rhodobaca bogoriensis investigated by electron microscopy

Electron microscopy and single-particle averaging were performed on isolated reaction centre (RC)—antenna complexes (RC–LH1–PufX complexes) of Rhodobaca bogoriensis strain LBB1, with the aim of establishing the LH1 antenna conformation, and, in particular, the structural role of the PufX protein. Projection maps of dimeric complexes were obtained at 13 Å resolution and show the positions of the 2 × 14 LH1 α- and β-subunits. This new dimeric complex displays two open, C-shaped LH1 aggregates of 13 αβ polypeptides partially surrounding the RCs plus two LH1 units forming the dimer interface in the centre. Between the interface and the two half rings are two openings on each side. Next to the openings, there are four additional densities present per dimer, considered to be occupied by four copies of PufX. The position of the RC in our model was verified by comparison with RC–LH1–PufX complexes in membranes. Our model differs from previously proposed configurations for Rhodobacter species in which the LH1 ribbon is continuous in the shape of an S, and the stoichiometry is of one PufX per RC.     

Evolution of low-light adapted peripheral light-harvesting complexes in strains of Rhodopseudomonas palustris

Purple bacteria have peripheral light-harvesting (PLH) complexes adapted to high-light (LH2) and low-light (LH3, LH4) growth conditions. The latter two have only been fully characterised in Rhodopseudomonas acidophila 7050 and Rhodopseudomonas palustris CGA009, respectively. It is known that LH4 complexes are expressed under the control of two light sensing bacteriophytochromes (BphPs). Recent genomic sequencing of a number of Rps. palustris strains has provided extensive information on PLH genes. We show that both LH3 and LH4 complexes are present in Rps. palustris and have evolved in the same operon controlled by the two adjacent BphPs. Two rare marker genes indicate that a gene cluster CL2, containing LH2 genes and the BphP RpBphP4, was internally transferred within the genome to form a new operon CL1. In CL1, RpBphP4 underwent gene duplication to RpBphP2 and RpBphP3, which evolved to sense light intensity rather than spectral red/far-red intensity ratio. We show that a second LH2 complex was acquired in CL1 belonging to a different PLH clade and these two PLH complexes co-evolved together into LH3 or LH4 complexes. The near-infrared spectra provide additional support for our conclusions on the evolution of PLH complexes based on genomic data.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.