THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

The N-terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo

The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N-terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N-terminal extension is needed for correct intracellular sorting in vivo , translational fusions were constructed of the translocator cDNA—with and without presequence—with the β-glucuronidase ( gus ) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo .     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.     

Distributions of beta sheets in proteins with application to structure prediction

We recently developed the Rosetta algorithm for ab initio protein structure prediction, which generates protein structures from fragment libraries using simulated annealing. The scoring function in this algorithm favors the assembly of strands into sheets. However, it does not discriminate between different sheet motifs. After generating many structures using Rosetta, we found that the folding algorithm predominantly generates very local structures. We surveyed the distribution of beta-sheet motifs with two edge strands (open sheets) in a large set of non-homologous proteins. We investigated how much of that distribution can be accounted for by rules previously published in the literature, and developed a filter and a scoring method that enables us to improve protein structure prediction for beta-sheet proteins. Proteins 2002;48:85-97.     

VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10⁻¹⁰ cm²/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.