SNARE Proteins-Why So Many,Why So Few?

Abstract: Both trafficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a small number of proteins, i.e., the exocytic core complex. In nerve terminals, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP-25 at the plasma membrane. In this survey, different secretory systems that depend on the exocytic core proteins are considered. The possibility that specificity in membrane recognition and fusion is achieved by the numerous variants of proteins of the exocytic core is discussed. Variability of the core complex proteins is determined by the complexity of gene families, isoform-specific localization, and posttranslational modifications. Basic biochemical properties depend on specific isoforms, and the possible protein-protein interactions are determined, in turn, by the compatibility of different isoforms. A correlation between specific variants and distinct biochemical or cellular properties is shown. The outcome of this survey is that heterogeneity in secretion may be dictated by the large number of possible combinations of variants of only a few proteins.     

The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts

Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3′ ends of mRNA. Using the two-hybrid system, we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1. Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway. These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system. The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1. Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis. Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing. These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity. Received: 6 January 1997 / Accepted: 27 February 1997     

Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media.

Seven chemicals, three buffers, and a salt solution known to affect bacterial attachment were tested to quantify their abilities to enhance the penetration of Alcaligenes paradoxus in porous media. Chemical treatments included Tween 20 (a nonionic surfactant that affects hydrophobic interactions), sodium dodecyl sulfate (an anionic surfactant), EDTA (a cell membrane permeabilizer that removes outer membrane lipopolysaccharides), sodium PPi (a surface charge modifier), sodium periodate (an oxidizer that cleaves surface polysaccharides), lysozyme (an enzyme that cleaves cell wall components), and proteinase K (a nonspecific protease that cleaves peptide bonds). Buffers included MOPS [3-(N-morpholino)propanesulfonic acid], Tris, phosphate, and an unbuffered solution containing only NaCl. Transport characteristics in the porous media were compared by using a sticking coefficient, alpha, defined as the rate at which particles stick to a grain of medium divided by the rate at which they strike the grain. Tween 20 reduced alpha by 2.5 orders of magnitude, to alpha = 0.0016, and was the most effective chemical treatment for decreasing bacterial attachment to glass beads in buffered solutions. Similar reductions in alpha were achieved in unbuffered solutions by reducing the solution ionic strength to 0.01 mM. EDTA, protease, and other treatments designed to alter cell structures did not reduce alpha by more than an order of magnitude. The number of bacteria retained by the porous media was decreased by treatments that made A. paradoxus more hydrophobic and less electrostatically charged, although alpha was poorly correlated with electrophoretic mobility and hydrophobicity index measurements at lower alpha values.(ABSTRACT TRUNCATED AT 250 WORDS)     

The preservation of Mycoplasma capricolum cell intactness after phospholipase A2 treatment

(1) By treating Mycoplasma capricolum cells with phospholipase A₂ about 80% of membrane phospholipids were rapidly hydrolyzed. The rate and extent of hydrolysis (at 37°C) were the same in intact cells and in isolated unsealed membranes. (2) Due to the low endogenous lysophospholipase activity detected in M. capricolum, phospholipase A₂ treatment resulted in the accumulation of lysophospholipids and free fatty acids. The free fatty acids were efficiently extracted from the cells by 1% bovine serum albumin whereas the lysophospholipids were almost fully retained within the cell membrane. (3) Following phospholipase A₂ treatment in the presence of 1% bovine serum albumin, cell intactness was preserved as indicated by the constant absorbance of the cell suspension and the retention of nucleic acids and NADH dehydrogenase activity within the cells. The treated cells showed, however, a slight decrease in K⁺ content and a decrease in cell viability. Viability was fully preserved after phospholipase A₂ treatment of cells grown with exogenous sphingomyelin. (4) Adapting M. capricolum to a cholesterol-poor medium resulted in a marked decrease in the cholesterol to phospholipid molar ratio (from about 1.1 to 0.3). Phospholipase A₂ treatment of the cholesterol-poor cells resuted in cell lysis. Cell lysis was induced in the cholesterol-rich cells by hydrolysing the lysophospholipids accumulated following phospholipase A₂ treatment. (5) It is suggested that after phospholipase A₂ treatment of M. capricolum cells, a relatively stable cell membrane is maintained and cell intactness is preseved due to the interaction of cholesterol, present in high amount in this membrane, with the lysophospholipids formed.     

Ribosyl-diphthamide: Confirmation of structure by fast atom bombardment mass spectrometry

Diphtheria toxin inactivates protein synthesis elongation factor 2 by attaching ADP-ribose to an unusual post-translational amino acid derivative, diphthamide, in the factor. Previously, we prepared ribosyl-diphthamide from the ADP-ribosyl-factor and proposed on the basis of NMR spectral analysis that it is 1-α-d-ribofuranosyl-2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine [N. J. Oppenheimer, and J. W. Bodley, (1981) J. Biol. Chem.256, 8579–8581 and op. cit.]. Now, using fast atom bomardment mass spectrometry, the intact cation of ribosyl-diphthamide has been observed in the gas phase. The theoretical mass of the structure proposed for ribosyl-diphthamide uniquely agrees with the observed mass of the inact cation of the compound to within 2 ppm. Collisional activation decomposition mass spectral analysis provided additional structural confirmation. Thus, although the compound has not been synthesized, all available evidence appears uniquely consistent with the structure of ribosyl-diphthamide previously proposed.     

Multiple ribosome binding to the 5'-terminal leader sequence of tobacco mosaic virus RNA. Assembly of an 80S ribosome X mRNA complex at the AUU codon

Tobacco mosaic virus (TMV) RNA with a long 5'-terminal leader sequence, as well as its isolated leader fragment (called omega), can form disome initiation complexes with wheat germ ribosomes. The second ribosome of the disome complex is bound to the leader sequence, upstream of an 80S particle occupying the AUG-containing initiation site [ Filipowicz and Haenni (1979) Proc. Natl Acad. Sci. USA 76, 3111-3115; Konarska et al. (1981) Eur. J. Biochem. 114, 221-227]. In order to identify the parts of omega important for interaction with ribosomes, the 5'-terminally-labelled omega was treated with alkali and the resultant fragments of different lengths were used in binding experiments. A 16-nucleotide-long fragment bearing the AUU sequence at the 3' end is the shortest oligonucleotide capable of forming 80S complexes with wheat germ ribosomes. Full-length (73 nucleotides) omega with AUG at the 3' terminus is the only RNA fragment supporting disome complex formation. Synthetic oligoribonucleotides were prepared for a study of 80S complex assembly at codons other than AUG. Hexadecanucleotide (A) 13A -U-U and, to lesser extent, also (A) 13A -U-C, (A) 13A -U-A and (A) 13A -C-G bind 80S ribosomes. Formation of the (A) 13A -U-U X 80S complex is dependent on the presence of initiator Met- tRNAMerf . Assembly of the 80S particle at the AUU sequence is not an artifact resulting from the terminal position of this triplet. (A) 13A -U-U elongated with over 100 A residues still efficiently binds an 80S ribosome positioned, as established by ribosome protection experiments, at the AUU triplet. The present results support the notion that 80S initiation-like complexes can be formed at sequences containing AUU codons. The possible function of these complexes as intermediates in initiation of translation of some viral RNAs is discussed.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.