RanGAPI is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state. Here, we report the amino acid sequence of RanGAPI, derived from cDNA and peptide sequences. We found it to be homologous to murine Fugl, implicated in early embryonic development, and to Rnalp from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mutations of budding yeast RNA] are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport. Concurrently, we have isolated Rnalp as the major RanGAP activity from Sc. pombe. Both this protein and recombinant Rnalp were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology. The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rnalp is reported to be localized to the cytoplasm. We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound

The primary structure ofporcine brain (3-tubulin was determined by automated and manual Edman degradation of six sets of overlapping peptides. The protein consists of 445 amino acid residues and has a minimum of six positions that are heterogeneous, indicating at least two (3-tubulins in porcine brain. Com-parison of the optimally aligned sequences of a-tubulin and 13-tubulin indicates that 41% of their primary structures are identical. A region rich in glycyl residues is similar both in sequence and predicted secondary structure to the phosphate binding loop of several nucleotide binding enzymes. fi-Tubulin contains a highly acidic COOH-terminal region that resembles the NH2-terminus of troponin T.Microtubules are helical arrays of alternating globular units of a-tubulin and 83-tubulin, each having a molecular weight of 50,000, a similar amino acid composition, and a similar overall shape (for review, see ref. 1). Yet these subunits seem to differ in function, as indicated by the binding of exchangeable GTP to f3-tubulin (2) and by the participation of a tissue-specific /3-tubulin in the meiotic spindle and other stages of spermatogenesis in Drosophila (3, 4). P-Tubulin mutants have been shown to cause reduced binding of cytostatic drugs (5,6) and were used to demonstrate the involvement of microtubules in nuclear movement in germinating spores of Aspergillus (7). A knowledge of the primary structure of ,3-tubulin, along with that of a-tubulin, could be helpful in understanding how different tubulins function, how the assembly of microtubules into various organelles is regulated, and how certain drugs and nucleotides are bound with high affinity. The sequence of a-tubulin has already shown several variants of this protein in brain (8). Regional similarities were found to muscle proteins, indicating the possibility ofcommon biochemical features in muscular and microtubular movement. Here we present the sequence of l3-tubulin from porcine brain and compare it with that of the a-chain. MATERIALS AND METHODSTubulin was purified from porcine brain as described (8). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer, pH 7.0, was chromatographed on DEAE-cellulose with a linear gradient of0. 1-0. 3 M sodium chloride. The protein was reduced and alkylated with iodoacetic acid and assayed for contaminating proteins by disc gel electrophoresis in the system of Yang and Criddle (9) using 8% polyacrylamide gels. a-and ,/3chains were separated on hydroxylapatite in 0.1% NaDodSO4 with a linear gradient of 0.2-0.4 M sodium phosphate (10). Fractions were assayed for purity by gel electrophoresis as above. Only }3-chain preparations containing <5% impurities were used for sequence determination.To remove NaDodSO4, the protein was dialyzed against 1 mM ammonium bicarbonate, then, the solution was concentrated by vacuum evaporation and brought to pH 5.5 with acetic acid, and the protein was precipitated with 9 vol of ice-cold acetone. The supernatant was discarded after 2 hr at -200C, and the precipit...

The amino acid sequence of a-tubulin from porcine brain was determined by automated and manual Edman degradation of eight sets of overlapping peptides. It comprises 450 residues plus a COOH-terminal tyrosine that is present only in 15% of the material. A region of 40 residues at the COOH-terminus is highly acidic, mainly due to 16 glutamyl residues. This high concentration ofnegative charge suggests a region for binding cations. At least six positions, most of them around position 270, are occupied by two amino acid residues each. Several of these exchange sites were assigned to specific peptides by analysis of the purified corresponding fragments. These data indicate four a-tubulins in porcine brain. Although a-tubulin on the whole is unrelated to other proteins, there are regions that can be correlated to sequences of the myosin head, to actin, to tropomyosin, and to troponins C and T.Tubulins occur in all eukaryotic cells as the constituents of microtubules, which participate in cell division, intracellular transport and secretion processes, ciliary and flagellar movement, morphogenesis, and cell orientation. Tubulins from widely differing species and cell types appear to be remarkably similar regarding composition, molecular weight, binding of cytostatic and psychopharmacological drugs, immunological crossreactivity, and capacity to copolymerize. Yet even within one cell, there are several types of microtubules that have differing stabilities and assemble into distinct organelles at various times. Knowledge of the primary structure should clarify whether there is just one tubulin for all functions or whether there exists a family of similar proteins. It will also facilitate mapping of binding sites for various ligands, production of antibodies to well-defined antigenic sites, matching of protein structure with that of messengers and genes, and investigation of functionally defective tubulin mutants. Comparison of the structure with those of known proteins may give hints for experiments regarding tubulin function.Tubulin in solution is assumed to exist as a heterodimer of two chains, a and f, each with a molecular weight4of 50,000, and very similar amino acid compositions. Yet functional differences have been reported. For example, only a-tubulin (from blood platelets) binds cyclic AMP (1) and only /3-tubulin binds exchangeable GTP (2). Here we present the sequence of the a-chain from porcine brain and report on the general strategy used. MATERIALS AND METHODSWe have purified tubulin from porcine brain by a modification of the methods used by Eipper (3) and by Luduena et al. (4). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer (pH 7.0) was incubated with 0.1 mM colchicine for 15 min at 37°C before chromatography on DEAE-cellulose with a linear gradient of 0.1-0.3 M sodium chloride. Tubulin was identified by the fluorescence of its complex with colchicine (5). The preparation was reduced, alkylated with iodoacetic acid, and assayed for protein impurities by disc gel electrophore...

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O 6 -methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids. P rimary or acquired antitumor therapy resistance is one of the major obstacles in oncology. For glioma, to date, this is pivotal for the standard of care, radiotherapy, and temozolomide (TMZ) alkylating chemotherapy. The DNA repair protein O 6