Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.

Nicotine is the major addictive agent in tobacco smoke, and it is metabolized extensively by oxidation and glucuronide conjugation. The contributions of ethnicity and UGT2B10 haplotype on variation in nicotine metabolism were investigated. Nicotine metabolism was evaluated in two populations of smokers. In one population of African American and European American smokers (n ϭ 93), nicotine and its metabolites were analyzed in plasma and 24-h urine over 3 days while participants were abstinent and at steady state on the nicotine patch. In a second study of smokers (n ϭ 84), the relationship of a UGT2B10 haplotype linked with D67Y to nicotine and cotinine glucuronidation levels was determined. We observed that both African American ethnicity and the UGT2B10 D67Y allele were associated with a low glucuronidation phenotype. African Americans excreted less nicotine and cotinine as their glucuronide conjugates compared with European Americans; percentage of nicotine glucuronidation, 18.1 versus 29.3 (p Ͻ 0.002) and percentage of cotinine glucuronidation, 41.4 versus 61.7 (p Ͻ 0.0001). In smokers with a UGT2B10 Tyr67 allele, glucuronide conjugation of nicotine and cotinine was decreased by 20% compared with smokers without this allele. Two key outcomes are reported here. First, the observation that African Americans have lower nicotine and cotinine glucuronidation was confirmed in a population of abstinent smokers on the nicotine patch. Second, we provide the first convincing evidence that UGT2B10 is a key catalyst of these glucuronidation pathways in vivo.

Summary PKA holoenzymes contain two catalytic (C) and a regulatory (R) subunit dimer where the two R-subunits are linked by an N-terminal Dimerization/Docking (D/D) domain. Cooperative binding of four cAMP molecules induces major structural changes in the R-subunits that cause kinase activation. While cooperativity exists between the two tandem cAMP binding domains, additional levels of cooperativity are associated with the tetramer. This allostery cannot be appreciated by studying heterodimers formed between C-subunit and deletion mutants of R that lack the D/D domain. Of critical importance is the flexible linker in the R-subunit that contains the Inhibitor Site (IS) that mimics the PKA substrate sequence and binds to the active site of the C-subunit. Two flexible linkers connect the IS to the D/D domain (N-Linker) and to the cAMP binding domains (C-Linker). In the cAMP-bound conformation IS and the C-Linker are disordered but become structured at the R:C interface when RIα binds to the C-subunit. The overall conformation of the tetramer, however, is mediated in large part by the N-Linker. To probe the function of the N-Linker in RIα and specifically to determine how the N-Linker contributes to assembly of the tetrameric holoenzyme, we engineered a monomeric form of RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Our previous RIα constructs began with the inhibitor site. RIα(73-244):C holoenzyme does not form stable dimers in solution without the dimerization domain; however, part of the extended linker is now ordered by interactions with a symmetry-related-dimer in the crystal. This complex of two symmetry-related dimers forms a tetramer that not only reveals novel mechanisms for allosteric regulation but also has many features that are consistent with known properties of the full-length holoenzyme. A model of the tetrameric RIα holoenzyme, based on this structure, is also consistent with small angle X-ray and neutron scattering data reported earlier. The model has been validated with new SAXS data and with a mutant of RIα that is localized to a novel interface that is unique to the tetramer.

Biomarkers of carcinogen uptake could provide important information pertinent to the question of exposure to environmental tobacco smoke (ETS) in childhood and cancer development later in life. Previous studies have focused on exposures before birth and during childhood, but carcinogen uptake from ETS in infants has not been reported. Exposures in infants could be higher than in children or adults because of their proximity to parents who smoke. Therefore, we quantified 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL) in the urine of 144 infants, ages 3 to 12 months, who lived in homes with parents who smoked. Total NNAL is an accepted biomarker of uptake of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cotinine and its glucuronide (total cotinine) and nicotine and its glucuronide (total nicotine) were also quantified. Total NNAL was detectable in 67 of 144 infants (46.5%). Mean levels of total NNAL in the 144 infants were 0.083 F 0.200 pmol/mL, whereas those of total cotinine and total nicotine were 0.133 F 0.190 and 0.069 F 0.102 nmol/mL, respectively. The number of cigarettes smoked per week in the home or car by any family member when the infant was present was significantly higher (P < 0.0001) when NNAL was detected than when it was not (76.0 F 88.1 versus 27.1 F 38.2). The mean level of NNAL detected in the urine of these infants was higher than in most other field studies of ETS exposure. The results of this study show substantial uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in infants exposed to ETS and support the concept that persistent ETS exposure in childhood could be related to cancer later in life. (Cancer Epidemiol Biomarkers Prev 2006;15(5):988 -92)

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Protein kinase A holoenzyme contains two Catalytic (C) and two Regulatory (R) subunits, which dimerize through their N‐terminus Dimerization/Docking (D/D) domains. R‐subunit isoforms are highly conserved; the most divergent region lies at the linker connecting the D/D domain and the two tandem cAMP binding domains. Binding of four cAMP molecules to the R‐subunit dimer leads to significant structural changes that lead to the activation of the kinase. This process is highly cooperative in the tetrameric holoenzyme but the cooperativity is significantly reduced in the heterodimer which lacks the D/D domain and the linker. To understand the role of the linker region, we solved the structure of a PKA RIα(73–244)/C heterodimer, which contains the first cAMP binding domain and most of the linker. The overall structure is very similar to the previously solved RIα(91–244)/C heterodimer complex, however, the new structure allowed observation of 7 more residues (84–90) in the linker. Interestingly, this linker region docks to another symmetry related heterodimer, suggesting that one R‐subunit not only binds to one C‐subunit at multiple sites within the heterodimer but also cross‐talks with another heterodimer through the linker region when the tetrameric complex is formed. The β4‐β5 loop of the R‐subunit and the Y235PPPFF239 of the C‐subunit, which links αF and αG and is where PKI binds are also involved in this cross‐talk. By integrating small angle X‐ray/neutron scattering, molecular interface free‐energy calculations and other data, a model of RIα/C tetrameric complex is proposed.

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