Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A 3 does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121-and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits. INTRODUCTIONThe possession of a nuclear envelope (NE) that encompasses the genome is the defining characteristic of all eukaryotes. The envelope consists of double nuclear membranes, hundreds to thousands of nuclear pore complexes (NPCs), and in higher eukaryotes, a nuclear lamina. Bidirectional transport of protein and RNA molecules through the nuclear envelope is mediated exclusively by NPCs, large structures ϳ60 -125 MDa in size (Reichelt et al., 1990;Macara, 2001;Quimby and Corbett, 2001;Goldfarb et al., 2004;Pemberton and Paschal, 2005;Patel et al., 2007).In higher eukaryotes the nuclear envelope, including pore complexes, disassembles at mitosis as a prelude to spindle assembly and chromosome segregation (Burke and Ellenberg, 2002;Margalit et al., 2005;Prunuske et al., 2006). This disassembly then necessitates nuclear envelope reformation around each set of segregated chromosomes toward the end of mitosis, a process that involves both nuclear membrane recruitment and nuclear pore formation.Analysis of the pore subunits produced by mitotic disassembly has provided the most useful clues to nearest neighbor interactions within the vertebrate pore. Vertebrate nuclear pores are comprised of ϳ30 different proteins or nucleoporins (Nups) in 8-32 copies each, to give a 500-1000 protein structure (Cronshaw et al., 2002). At mitosis the massive vertebrate pore disassembles into ϳ14 soluble subunits, each with a distinct protein composition, whereas the integral membrane pore proteins, POM121, NDC1, and gp210, segregate into endoplasmic reticulum (ER) sheets and vesicles (Gerace et al., 1982;Wozniak et al., 1989;Greber et al., 1990;Hallberg ...

Proton decoupled deuterium NMR spectra of oriented bilayers made of DMPC and 30 mol % deuterated cholesterol acquired at 76.8 MHz (30 degreesC) have provided a set of very accurate quadrupolar splitting for eight C-D bonds of cholesterol. Due to the new precision of the experimental data, the original analysis by. Biochemistry. 23:6062-6071) had to be reconsidered. We performed a systematic study of the influence on the precision and uniqueness of the data-fitting procedure of: (i) the coordinates derived from x-ray, neutron scattering, or force field-minimized structures, (ii) internal mobility, (iii) the axial symmetry hypothesis, and (iv) the knowledge of some quadrupolar splitting assignments. Good agreement between experiment and theory could be obtained only with the neutron scattering structure, for which both axial symmetry hypothesis and full order parameter matrix analysis gave satisfactory results. Finally, this work revealed an average orientation of cholesterol slightly different from those previously published and, most importantly, a molecular order parameter equal to 0.95 +/- 0.01, instead of 0.79 +/- 0.03 previously found for the same system at 30 degreesC. Temperature dependence in the 20-50 degreesC range shows a constant average orientation and a monotonous decrease of cholesterol Smol, with a slope of -0.0016 K-1. A molecular order parameter of 0.89 +/- 0.01 at 30 degreesC was determined for a DMPC/16 mol % of cholesterol.

Initial transport kinetics of isoniazid (INH) and its uptake a t the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x lom6 M to M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 "C) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity a t the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.