The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio VopL depends on its three W domains and dimerization through a unique VopL C-terminal domain (VCD). The VCD displays a novel all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment, but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

Abbreviations: LatB, latrunculin B; PRF1, Chlamydomonas reinhardtii profilin; FOR1, 39Chlamydomonas reinhardtii formin 1; PRR, proline rich region; TIRF, total internal 40 reflection fluorescence; G-actin, globular actin; F-actin, filamentous actin 41 42 SUMMARY STATEMENT 43The Chlamydomonas reinhardtii formin FOR1 initiates rapid assembly of fertilization 44 tubule actin filaments from monomers associated with the actin-assembly inhibitor profilin 45 PRF1. 46 47ABSTRACT 48 49The regulated assembly of multiple filamentous actin (F-actin) networks from an actin 50 monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii 51 is a unicellular green alga expressing a conventional and divergent actin that is an 52 emerging system for investigating the complex regulation of actin polymerization. One 53 actin network that contains exclusively conventional F-actin in Chlamydomonas is the 54 fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to 55 two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes 56 (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that 57 unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both 58 F-actin nucleation and barbed end elongation at equimolar concentrations to actin. 59However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-60 bound actin. PRF1 further favors FOR1-mediated actin assembly by potently inhibiting 61Arp2/3 complex-mediated actin assembly. Furthermore, for1 and prf1-1 mutants, as well 62 as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in 63 gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a 64 primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 65

An algal profilin PRF1 inhibits actin filament nucleation and elongation at low concentrations that can be overcome by the formin FOR1 for rapid actin assembly. FOR1 is required for assembly of a mating structure in gametic algal cells. Together, FOR1 and PRF1 cooperate to selectively assemble F-actin at the right time and place.