Plasma membrane specialization at the discharge site of the excretory poreless vacuole of the ciliate Euplotes raikovi

The contractile vacuole of Euplotes raikovi consists basically of a membrane-delimited cistern that has no direct communication with the exterior of the cell. Contiguous alveoli interpose between the cisterna and the plasma membrane and probably receive the cisternal contents before being expelled. The area of the plasma membrane that interacts with the outer membrane of the alveoli overlying the cistern has been found to carry a special region of intramembranous particles organized in 100-200 strands, showing varied sizes and a tendency to align parallel with one another. The region is also characterized by a smooth central core and by characteristic deformations that have been interpreted as focal sites of discharge of the contractile vacuole.     

A 20 kDa erythrocyte membrane phosphoprotein

A small (approximately 20 kDa) protein associated with the human erythrocyte membrane undergoes phosphorylation that is potentiated by the addition of phosphatidylinositol. It is indicated that phosphatidylinositol 4,5-bisphosphate, generated in situ during the protein phosphorylation reaction, is the effective agent. Phosphorylation of the small protein is also potentiated when isolated membranes are exposed to conditions that promote the phosphorylation of the phosphatidylinositols. It is suggested that the 20 kDa membrane protein participates in biochemical signaling in the erythrocyte.Abbreviations EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Growth of Crithidia at High Temperature: Crithidia hutneri sp. n. and Crithidia luciliae thermophila s. sp. n.*

SYNOPSIS. Crithidia hutneri sp. n. and Crithidia luciliae thermophila s. sp. n. are described. Both flagellates can be grown in a defined medium over a temperature range of 15–37°C. The requirements for amino acids, vitamins, purine and hemin, and pH range were similar to those established for Crithidia fasciculata, although threonine was required as a growth factor for C. luciliae thermophila at high temperatures. Adenosine could be used by the 2 Crithidia as a purine source at 28 but not at 37 C.     

Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.     

The unique N‐terminal insert in the ribosomal protein,phosphoprotein P0, of Tetrahymena thermophila: Bioinformatic evidence for an interaction with 26S rRNA

Phosphoprotein P0 (P0) is part of the stalk complex of the eukaryotic large ribosomal subunit necessary for recruiting elongation factors. While the P0 sequence is highly conserved, our group noted a 15–16 residue insert exclusive to the P0s of ciliated protists, including Tetrahymena thermophila. We hypothesized that this insert may have a function unique in ciliated protists, such as stalk regulation via phosphorylation of the insert. Almost no mention of this insert exists in the literature, and although the T. thermophila ribosome has been crystallized, there is limited structural data for Tetrahymena's P0 (TtP0) and its insert. To investigate the structure and function of the TtP0 insert, we performed in silico analyses. The TtP0 sequence was scanned with phosphorylation site prediction tools to detect the likelihood of phosphorylation in the insert. TtP0's sequence was also used to produce a homology model of the N‐terminal domain of TtP0, including the insert. When the insert was modeled in the context of the 26S rRNA, it associated with a region identified as expansion segment 7B (ES7B), suggesting a potential functional interaction between ES7B and the insert in T. thermophila. We were not able to obtain sufficient data to determine whether a similar relationship exists in other ciliated protists. This study lays the groundwork for future experimental studies to verify the presence of TtP0 insert/ES7 interactions in Tetrahymena, and to explore their functional significance during protein synthesis. Proteins 2015; 83:1078–1090. © 2015 Wiley Periodicals, Inc.     

A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases

When the plasma membranes of caput and cauda epididymal spermatozoa of hamster were evaluated for their ability to undergo phosphorylation, a differential phosphorylation of the membrane proteins was observed. In the plasma membranes of the caput epididymal spermatozoa (immature), twelve proteins were phosphorylated (100, 76, 67, 65, 55, 52, 47, 42, 38, 32, 30, and 20 kD), whereas in the plasma membranes of cauda epididymal spermatozoa (mature), a differential phosphorylation pattern was observed with respect to the 94, 67, 52, and 47 kD proteins. The 94 kD protein was found to be phosphorylated and the 67 kD protein was found to be not phosphorylated in cauda spermatozoal plasma membrane (Cd SPM) in contrast to this protein in caput spermatozoal plasma membrane (Cpt SPM). The 52 and 47 kD proteins were also more intensely phosphorylated in Cd SPM than Cpt SPM. The 100 kilodalton protein, although present in both Cpt and Cd sperm plasma membranes, was found to be phosphorylated at the tyrosine residues only in the Cd SPM, as indicated by the Western blot using antiphosphotyrosine antibody. Further, a differential phosphorylation of the substrate proteins present in the Cpt and Cd SPM was seen when Mg²⁺ in the assay buffer was replaced by other divalent cations. For instance, Zn²⁺ stimulated the phosphorylation of an 85 kD protein in cauda SPM and not in the caput SPM and Ca²⁺ stimulated the phosphorylation of a 76 kD protein only in the cauda SPM. The phosphoproteins in both the plasma membranes were found to be phosphorylated predominantly at the tyrosine residue. The differential phosphorylation of a 100 kD protein at tyrosine in the Cd SPM (Western blot), which is absent in the immature Cpt SPM, also indicated that certain proteins in the hamster spermatozoa are phosphorylated in a maturation-specific manner. Mol. Reprod. Dev. 47:341–350, 1997. © 1997 Wiley-Liss, Inc.     

Nature of the phosphorylated 26 000 molecular weight protein extracted from dog thyroid with contractile proteins

Dog thyroid contractile proteins are characterized by their ATPase activity at high KCl concentration. In the presence fo Ca²⁺, 80 nmol ATP are hydrolyzed per min per mg protein. This Ca²⁺-ATPase activity is inhibited by Mg²⁺ but not influenced by sodium azide.The 26 000 molecular weight protein which is present in thyroid contractile protein preparations and the phosphorylation of which is stimulated by thyroid stimulating hormone (TSH) is suggested to be identical to the lysine-rich histones (H₁). Indeed, radioactive thyroid H₁ histones added to unlabelled thyroid slices copurify with the contractile proteins and migrate at the same level as the 26 000 molecular weight protein when submitted to electrophoresis in polyacrylamide sodium dodecyl sulfate gels of different acrylamide concentrations.     

Correlation of structure and function of chloroplast membranes at the supramolecular level

Freeze-fracture electron microscopy has revealed that different size classes of intramembrane particles of chloroplast membranes are nonrandomly distributed between appressed grana and nonappressed stroma membrane regions. It is now generally assumed that thylakoid membranes contain five major functional complexes, each of which can give rise to an intramembrane particle of a defined size. These are the photosystem II complex, the photosystem I complex, the cytochrome f/b6 complex, the chlorophyll a/b light-harvesting complex, and the CF0 -CF1 ATP synthetase complex. By mapping the distribution of the different categories of intramembrane particles, information on the lateral organization of functional membrane units of thylakoid membranes can be determined. In this review, we present a brief summary of the evidence supporting the correlation of specific categories of intramembrane particles with known biochemical entities. In addition, we discuss studies showing that ions and phosphorylation of the membrane adhesion factor, the chlorophyll a/b light-harvesting complex, can affect the lateral organization of chloroplast membrane components and thereby regulate membrane function.     

Identification of goat sperm ecto-cyclic AMP independent protein kinase substrate localized on sperm outer surface

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.     

Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open

The human malaria parasite Plasmodium falciparum resides and multiplies within a membrane-bound vacuole in the cytosol of its host cell, the mature human erythrocyte. To enable the parasite to complete its intraerythrocytic life cycle, a large number of parasite proteins are synthesized and transported from the parasite to the infected cell. To gain access to the erythrocyte, parasite proteins must first cross the membrane of the parasitophorous vacuole (PVM), a process that is not well understood at the mechanistic level. Here, we review past and current literature on this topic, and make tentative predictions about the nature of the transport machinery required for transport of proteins across the PVM, and the molecular factors involved.     

A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.     

A study of the membrane-water interface region of membrane proteins

The most conspicuous structural characteristic of the alpha-helical membrane proteins is their long transmembrane alpha-helices. However, other structural elements, as yet largely ignored in statistical studies of membrane protein structure, are found in those parts of the protein that are located in the membrane-water interface region. Here, we show that this region is enriched in irregular structure and in interfacial helices running roughly parallel with the membrane surface, while beta-strands are extremely rare. The average amino acid composition is different between the interfacial helices, the parts of the transmembrane helices located in the interface region, and the irregular structures. In this region, hydrophobic and aromatic residues tend to point toward the membrane and charged/polar residues tend to point away from the membrane. The interface region thus imposes different constraints on protein structure than do the central hydrocarbon core of the membrane and the surrounding aqueous phase.     

Phosphorylation of phospholipase B in the plasma membrane of Saccharomyces cerevisiae

Abstract Plasma membrane vesicles from Saccharomyces cerevisiae were incubated with [γ-³²P]ATP. Several phosphorylated protein bands were separated by LiDS polyacrylamide gel electrophoresis. One of these bands with an apparent M _r of 145 000 was identified by immunoprecipitation as a membrane-bound phospholipase.     

Fast track to a phosphoprotein sketch - MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity

Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.     

Immunomodulating effects of Toxoplasma gondii occur at an early stage of infection

Abstract An antibody to a 60-kDa protein associated with membrane-bound ribosomes (MBRP) in Staphylococcus aureus was shown to cross-react with a 64-kDa protein in Bacillus subtilis . Evidence is presented suggesting that this Bacillus protein is identical to a 64-kDa protein, possibly involved in protein secretion, described by Horiuchi et al. [Proc. Natl. Acad. Sci. USA 80 (1983) 3287–3291). Both the 60-kDa staphylococcal protein and the 64-kDa Bacillus protein were precipitated as a complex with three other proteins when immunoprecipitated with the staphylococcal MBRP antibody.     

The human orthologue of CdGAP is a phosphoprotein and a GTPase-activating protein for Cdc42 and Rac1 but not RhoA

BACKGROUND INFORMATION: Rho GTPases regulate a wide range of cellular functions affecting both cell proliferation and cytoskeletal dynamics. They cycle between inactive GDP- and active GTP-bound states. This cycle is tightly regulated by GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Mouse CdGAP (mCdc42 GTPase-activating protein) has been previously identified and characterized as a specific GAP for Rac1 and Cdc42, but not for RhoA. It consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP-related genes are present in both vertebrates and invertebrates. We have recently reported that two predominant isoforms of CdGAP (250 and 90 kDa) exist in specific mouse tissues. RESULTS: In the present study, we have identified and characterized human CdGAP (KIAA1204) which shares 76% sequence identity to the long isoform of mCdGAP (mCdGAP-l). Similar to mCdGAP, it is active in vitro and in vivo on both Cdc42 and Rac1, but not RhoA, and is phosphorylated in vivo on serine and threonine residues. In contrast with mCdGAP-l, human CdGAP interacts with ERK1/2 (extracellular-signal-regulated kinase 1/2) through a region that does not involve a DEF (docking site for ERK Phe-Xaa-Phe-Pro) domain. Also, the tissue distribution of CdGAP proteins appears to be different between human and mouse species. Interestingly, we found that CdGAP proteins cause membrane blebbing in COS-7 cells. CONCLUSIONS: Our results suggest that CdGAP properties are well conserved between human and mouse species, and that CdGAP may play an unexpected role in apoptosis.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

Semen evaluation of giant pandas (Ailuropoda melanoleuca) at the Wolong Reserve

Semen from 4 wild-caught giant pandas (Ailuropoda melanoleuca) held at the China Conservation and Research Centre for the Giant Panda was collected (22 samples during 1991–1993) by electroejaculation, and evaluated for use in artificial insemination. Semen characteristics (mean ± SD) recorded were as follows: semen volume–1.5 ± .9 ml (range—0.3–3.5); sperm density 1.5 ± 0.1 × 10⁹/ml (range—0.24–4.2); motility 79 ± 10% (range—60–95); abnormal sperm 14 ± 5% (range—10–27); and pH 7.1 ± 0.2 (range—6.7–7.5). There were significant differences from year to year (P < 0.05) in semen volume collected and in the percentage abnormal sperm in 1993 compared to other years. There were no significant differences among semen produced from the four different pandas. Data collected were similar to reports for other giant pandas, and semen from all 4 giant pandas was considered suitable for use in artificial insemination. © 1994 Wiley-Liss, Inc.     

The synthesis of phaseolin: a model for the study of the plant secretory pathway

Abstract

Phascolin, the major seed storage protein of common bean (Phaseolus vulgaris), has been for many years one of the main working horses for studying protein synthesis, trafficking and structural maturation in the secretory pathway of higher plants. Recently, phaseolin has been used as a tool to determine molecular interactions between chaperones and newly-synthesised wild-type or structurally-defective secretory proteins in plant cells. Despite the vast amount of information available on the structure and the cell biology of phaseolin, the determinants for its sorting to the vacuole are still unknown.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.