The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein.

The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP). The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1. Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate). Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns. Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea. Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane. Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals. Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction. The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed.

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro.

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis.

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

Cell body responses to axonal injury: traumatic axotomy versus toxic neuropathy.

To compare the evolution of cell body responses to two different types of axonal injuries--sciatic nerve crush (axotomy) and chronic 2,5-hexanedione-induced neuropathy--we studied rat lumbar dorsal root ganglion neurons with light microscopy and morphometry. Compared with control neurons, axotomized cells showed early (1 day) increases in the frequencies of two responses, nuclear eccentricity and Nissl body displacement, and later (4 day) increases in average satellite cell nuclei and decreases in perikaryal diameters. In toxin-induced axonal degeneration, there were similar patterns of defined alterations, although the evolution progressed over weeks and the response magnitudes were smaller. We conclude that the two experimental conditions show basic morphologic similarities, implying cell body reorganization in toxic axonopathy may be a response to axonal dysfunction or degeneration.