E. coli incorporates increasing proportions of saturated and long-ehain fatty acids into phospholipids aS growth temperature is increased. It was found that this compositional variation results in the biosynthesis of phospholipids that have identical viscosities at the temperature of growth of the cells. This "homeoviscous adaptation" can also be observed in E. coli membrane preparations. Viscosities were determined by use of the electron spin resonance spin-label technique.Escherichia coli varies the fatty-acid composition of membrane lipids as a function of temperature of growth-as do many other organisms (1). As cells are grown at increasing temperatures, there is an increasing tendency to incorporate longer and more saturated fatty acids into phospholipids (2).One expected result of such a variation is that the phospholipids synthesized at progressively higher growth temperatures should exhibit progressively higher solid-to-liquid-crystalline phase transitions (3) and, indeed, an effect of this type has been reported (4). However, the true adaptive significance of the temperature-control phenomenon might be best revealed by examination of the physical state of the membrane lipids at the temperature of growth of the organism. In this report the fluidity of E. coli lipids and membranes is examined at the temperature of growth of the organism by use of an electron spin resonance (ESR) spin label. The results of this study are consistent with the hypothesis that variation of fatty-acid composition of membrane phospholipids serves in producing membranes whose lipids have a constant fluidity at the temperature of growth-a process dubbed "homeoviscous adaptation." Spin-labeled phospholipid samples were prepared by evaporating 0.4 ml of the spin label stock solution, giving a lipidto-probe ratio in the sample of 67:1 (wt/wt). MATERIALS AND METHODS Strains andThe mix was applied to 0.1 X 4 X 60-mm glass plates and the solvent allowed to evaporate. A second glass plate was then placed on top of the first and the edges were sealed with beeswax. The sample was then attached to a wooden applicator stick by means of Duco cement, providing the handle that was used to lower the sample into the ESR cavity.E. coli membranes were prepared by the method of Kaback (9). Membranes were taken up in the minimum possible volume of 0.05 M potassium phosphate buffer, pH = 7.0. (10)(11)(12) in terms of the rotational correlation time, x, for the system.* As used by Rich (14) for nitroxides, this equation takes the form:where h(0), h(1), and h(-1) are the amplitudes of the center, low-field and high-field lines, respectively, and AH (0)
Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LA⌬50/progerin). These modifications cause an abnormal association of LA⌬50/ progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LA⌬50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.cell division ͉ nuclear lamins ͉ nuclear structure ͉ progeria ͉ protein farnesylation
The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their early replication arrest. Treatment of patient cells with a protein farnesyltransferase inhibitor (FTI) did not result in reduction of DNA double-strand breaks and damage checkpoint signaling, although the treatment significantly reversed the aberrant shape of their nuclei. This suggests that DNA damage accumulation and aberrant nuclear morphology are independent phenotypes arising from prelamin A accumulation in these progeroid syndromes. Since DNA damage accumulation is an important contributor to the symptoms of HGPS, our results call into question the possibility of treatment of HGPS with FTIs alone.
The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p2lr", which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteinealiphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatintreated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.The nuclear lamina is a polymeric protein structure that lines the inner nuclear membrane. In most mammalian cells, it consists of three major proteins, lamins A-C (for review, see ref. 1). Mature lamin A is synthesized from a larger precursor protein (2, 3) and lacks the carboxyl-terminal 18 amino acids predicted by the cDNA sequence (4). Conversion ofthe lamin A precursor, prelamin A, to lamin A is dependent upon the isoprenylation of prelamin A (5). Prelamin A is an example of a class of proteins terminating in the sequence cysteinealiphatic-aliphatic-other (CAAX), which is prenylated at the consensus cysteine (for reviews, see refs. 6 and 7). This motif, shared by such proteins as p215 and the a-type mating factor of Saccharomyces, entrains a series of posttranslational processing steps. These include prenylation at the CAAX cysteine with either a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid followed by proteolytic removal of the A-A-X amino acids and carboxyl methylation of the now terminal cysteine. Evidence has been reported that the B-type lamins also undergo these prenylationdependent processing reactions (8-10). However, specific steps for the processing of prelamin A have not been directly demonstrated. Activities capable of catalyzing the carboxylterminal processing of a-factor and p2l's in vitro have been described (11,12). For p21'S, these activities were localized to the cytosolic and microsomal compartments (12, 13).Prelamin A and the yeast a-factor share an additional reaction subsequent to these carboxyl-terminal processing events. Both are subject to an endoproteolytic cleavage of their resp...
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