Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.
The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.
Microtubules are constituents of axonemes, mitotic spindles, and elaborate arrays in interphase cells, and, with intermediate filaments and microfilaments, are among the most prevalent structures visualized in the cytomatrix (22,44). With the exception ofthe A microtubule ofcilia and flagella, the lattice geometry of microtubules is highly conserved . However, each of the major subunits of microtubules, a-and a-tubulin, shows heterogeneity. The number ofa-and f3-tubulin subspecies differs among tissues and organisms, and a number of types of analysis are used to examine how these tubulin variants are related to specific cell functions (1, 9-11, 33, 40). Investigations of the number and complexity of genes coding for these polypeptides have also been initiated (see reference 13 for review). However, the mechanisms that regulate the posttranslational compartmentalization of subunits, the spatial and temporal assembly of subunits into microtubules, and the integration of microtubules in various cellular events are still largely unknown .There are many levels at which the formation and organization of microtubules might be determined. A postulate originating from early analyses of mitotic spindle formation (32) was that a pool of subunits existed in equilibrium with formed microtubules; increases in the subunit concentration could therefore result in a net increase in polymer. With few exceptions, however, a rapid increase in the total tubulin pool does not appear to occur before the elaboration of more extensive microtubule arrays . For example, our studies (42,50) have demonstrated that mouse neuroblastoma cells possessing microtubule-filled neurites contain four to five times more tubulin polymer than rounded, nondifferentiated cells, but the total tubulin content of these two cell types is the same. On the basis of volume calculations, the equilibrium concentration of subunits in the nondifferentiated cells is at least twice that in differentiated cells. Data such as this indicate that a simple equilibrium between subunit and polymer cannot account for the changes in microtubule formation coordinated with certain cellular events. In addition, recent findings show that an increase in the subunit concentration in cells, brought about either by drug treatment (15) or injection of tubulin (16), results in a depression of tubulin synthesis and the loss of tubulin mRNA. These data suggest that cells autoregulate the total tubulin pool and that this may be effected by "monitoring" of the monomer concentration (14).28s
INTRODUCTIONOver the last decade, a number of proteins have been identified that coassemble with microtubules in vitro. As outlined in this volume and elsewhere,I4 these include low molecular weight species from brain (tau), and high molecular weight proteins from brain ((microtubule-associated protein) MAP-1, MAP-2) and cultured cells (210 kD HeLa MAP, . Antisera against all of these MAPS stain interphase arrays in cultured cells, and most also react with mitotic spindles. Although studies on the distribution of these proteins in tissues are being initiated, the function of any of these MAPS in vivo is still largely unknown.We have been investigating the occurrence of MAP-43 in a variety of systems. This protein was originally identified as a 215 kD MAP in neuroblastoma cells, the synthesis of which appeared to be induced upon neurite differentiation.2s5 The distribution of MAP-4 in extracts3 and semi-thin sections6 of mouse tissues has recently been described. This paper outlines the species distribution of MAP-4, further analyses on the complexity of this MAP, and the occurrence of MAP-4 during brain development and in early mouse embryos. MATERIAL AND METHODSThe identification of MAP-4 in cells and tissues, the preparation of antisera to this MAP, and procedures used for immunoblotting and immunofluorescence have all been described previously.' Immunoelectron microscopy of microtubules formed in vivo was performed by fixing the samples with 1.0% glutaraldehyde in 0.1 M PIPES, pH 6.9, and placing the solutions on formvar and carbon-coated nickel grids. The grids were then inverted and treated at room temperature with the following solutions: phosphate buffered saline (PBS), 10 min; I mg/ml NaBH, in water, 15 min; 1% BSA in TBS, pH 7.6, 30 min; 1/20 MAP-4 or tubulin antibodies in TBS, pH 7.6 with 1% BSA, 1 hr; TBS, 1 mg/ml BSA, pH 8.3, 5 x 5 min rinses; goat anti-rabbit serum conjugated with 20 nm colloidal gold (1/20 in TBS, pH 8.3 with 1% BSA), 1 hr; TBS, pH 8.3, 5 x 5 'This work was supported by NIH Grant GM 22214 to J.B. Olrnsted. 292OLMSTED et af.: DISTRIBUTION OF MAP-4 293 min; and 1% glutaraldehyde in TBS, pH 8.3, 5 minutes. Grids were then rinsed successively with 3-4 drops each of water, 1 mg/ml cytochrome c, water, and 1% aqueous uranyl acetate, and air dried.Peptide mapping was carried out using a modification' of the method of Cleveland et uI.* Samples containing MAP-4 were resolved in a 15 cm 4-6% linear gradient acrylamide gel. A small portion of the lane was reserved for staining and an adjacent strip placed horizontally across a second SDS gel made with a 1.5 cm stacking gel (3.1% acrylamide) and a 14 cm running gel (1 2-1 8% linear gradient of acrylamide). The gel strip was overlaid with 0.5 ml sample buffer lacking mercaptoethanol and containing 0.5 pg/ml Stuphylococcus aureus V8 enzyme. Electrophoresis was carried out until the tracking dye reached the front of the stacking gel, and the current turned off for 30 min; electrophoresis was then continued until the tracking dye reached the bottom...
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