Histone-,B-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B. L. Roberts, W. D. Richardson, and A. E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to mjthionine abolished nuclear localization of an H2B-Igalactosidase fusion protein containing amino acids to 33 of H2B. However, an H2B-0-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.The biological importance of histones has led to a detailed knowledge of the primary sequence and functional domains of these proteins. The four core histones, H2A, H2B, H3, and H4, have a similar overall structure consisting of basic amino and carboxy termini and a globular hydrophobic internal region (for reviews, see references 23 and 33). The amino terminus of H2B is followed by a region of hydrophobic amino acids which has been identified as the minimal domain which interacts with H2A to form H2A-H2B dimers (amino acids 40 to 117 in Saccharomyces cere'iisiae H2B; 37, 51). H2B is the least conserved of the four core histones, with most of the variability occurring at the amino terminus. Amino acids 3 to 32 or 30 to 37 can be deleted from yeast H2B without impairment of cell growth (46, 52). These studies suggest either that there is no obligatory biological requirement for the H2B amino terminus or that the function(s) of this domain can be complemented by other histones in the nucleosome.During the period of yeast histone biosynthesis in late Gl and early S phase (20,22,39), approximately 4.0 x 105 histones per haploid genome are synthesized, transported into the nucleus, and assembled into nucleosomes. The number of histones which must be imported during the interval of DNA replication, as well as the requirement for their precise stoichiometry in nucleosome assembly, suggests that specific transport mechanisms are used in histone nuclear localization. I'he transport of proteins into the nucleus can result from either passive diffusion through nuclear pores (4,6,40,42) and accumulation in the nucleus by mass action (9) or by transport processes requiring recognition of an imported protein coupled to its active transport or facilitated diffusion (5, 10-12, 15, 19, 25, 26, 28, 29, 47). The existence of nuclear transport processes has been implicated in the nuclear localization of the Xenopus oocyte protein nucleoplasmin (11,12 to be required for nuclear uptake of pentameric nucleoplasmin oligomers (11). Moreover, gold particles as large as 20 nm which have been coated with nucle...

The identification of bacterial metabolites produced by the microbiota is a key point to understand its role in human health. Among them, lipo-amino acids (LpAA), which are able to cross the epithelial barrier and to act on the host, are poorly identified. Structural elucidation of few of them was performed by high-resolution tandem mass spectrometry based on electrospray combined with selective ion dissociations reach by collision-induced dissociation (CID). The negative ions were used for their advantages of yielding only few fragment ions sufficient to specify each part of LpAA with sensitivity. To find specific processes that help structural assignment, the negative ion dissociations have been scrutinized for an LpAA: the N-palmitoyl acyl group linked to glutamic acid (C16Glu). The singular behavior of [C16Glu-H]¯ towards CID showed tenth product ions, eight were described by expected fragment ions. In contrast, instead of the expected product ions due to CONH-CH bond cleavage, an abundant complementary dehydrated glutamic acid and fatty acid anion pair were observed. Specific to glutamic moiety, they were formed by a stepwise dissociation via molecular isomerization through ion–dipole formation prior to dissociation. This complex dissociated by partner splitting either directly or after inter-partner proton transfer. By this pathway, surprising regeneration of deprotonated fatty acid takes place. Such regeneration is comparable to that occurred from dissociation to peptides containing acid amino-acid. Modeling allow to confirm the proposed mechanisms explaining the unexpected behavior of this glutamate conjugate.

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