Herwig Ponstingl

Complete amino acid sequence of alpha-tubulin from porcine brain

The amino acid sequence of alpha-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 of negative 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 alpha-tubulins in porcine brain. Although alpha-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.

[17] Catalysis of guanine nucleotide exchange or Ran by RCC1 and stimulation of hydrolysis of Ran-bound GTP by Ran-GAP1

Publisher Summary Ran is a Ras-related protein found predominantly in the nucleus, with a conspicuous acidic carboxy-terminal sequence devoid of an attachment signal for prenylation, and therefore representing a separate family among the Ras-related proteins. It has been highly conserved in divergent species ranging from humans to protozoa, yeasts, and plants. Ran has been reported to be involved in the import of proteins with nuclear localization signals into the nucleus. It is not known how these functions are correlated or whether they reflect a single central aspect of Ran function. Strikingly, GTP hydrolysis appears to be required for these functions, judging from their impairment by Ran mutants deficient in GTP hydrolysis that were modeled after the permanently active form of Ras. Another prominent feature is the abundance of the main components of the RCC1–Ran pathway.

Human RanBP3, a group of nuclear RanGTP binding proteins

A group of novel human Ran‐binding proteins, RanBP3, was identified using the yeast two‐hybrid system via Ran‐mediated interaction with the nucleotide exchange factor RCC1. Several open reading frames, representing putative alternatively spliced products, were established by cDNA cloning. Two of them, RanBP3‐a and RanBP3‐b, encode nuclear hydrophilic proteins of 499 and 562 amino acid residues. The sequences contain FXFG motifs, characteristic of a subgroup of nucleoporins, and a C‐terminal domain showing similarity to the Ran‐binding protein RanBP1. These proteins are localized in the nucleus, preferentially bind RanGTP and may be nuclear effectors of the Ran pathway.

Tubulin sequence conservation

Various aspects of the primary structure of tubulin are discussed and tubulin sequence data are compared. A hypothesis concerning the evolution of tubulin is also presented. The main points raised are: (1) Identical glycyl rich regions in alpha- and beta-tubulin, which are similar in both sequence and predicted secondary structure to a region in several nucleotide binding enzymes, may be involved in binding GTP. (2) Small regions of homology are present to actin, myosin and troponin T. These homologous regions may have the same function, resulting in a convergence of their sequences, or they may have arisen by a pathway of protein evolution which is still only very poorly understood. (3) The mutation rate between pig and chick brain tubulin is 0.22 PAMs (accepted point mutations/100 residues) per hundred million years, which is comparable to that of the histones. At an early time in its history, however, the tubulin heterodimer appears to have had a relatively high rate of mutation. This may have been during the evolution of the first eukaryotes.

Loss of Drop1 expression already at early tumor stages in a wide range of human carcinomas.

In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20‐ to 180‐fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5′ region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two‐hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors.

Human Supt5h protein, a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis

The Saccharomyces cerevisiae proteins Spt4p, Spt5p and Spt6p are involved in transcriptional repression by modulating the structure of chromatin. From HeLa cells we have purified a human homologue of Spt5p, Supt5hp, and show here that the protein is reversibly phosphorylated in mitosis. The cloned cDNA predicts a protein of 1087 residues with 31% identity to yeast Spt5p. It includes an acidic N‐terminus, a putative nuclear localization signal and a C‐terminal region containing two different repeated motifs. One of them, with the consensus sequence P‐T/S‐P‐S‐P‐Q/A‐S/G‐Y, is similar to the C‐terminal domain in the largest subunit of RNA polymerase II.

DelGEF, a homologue of the Ran guanine nucleotide exchange factor RanGEF, binds to the exocyst component Sec5 and modulates secretion.

In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two‐hybrid system. The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.

A protease from Astacus fluviatilis as an aid in protein sequencing

Abstract A protease from the digestive tract of the crayfish Astacus fluviatilis (EC 3.4.99.6) which does not seem to belong to any of the known families of proteases, proves to be a valuable aid in sequencing proteins. In denatured polypeptide chains, it has the unusual property of hydrolyzing peptide bonds on the amino side of the small uncharged residues Ala, Thr, Ser, Gly, and Val. The number of cleavage sites, however, is restricted to about one-fifth of the respective residues present. A requirement for cleavage may be the capability of the polypeptide chain to assume a conformation similar to a reverse turn at the site of hydrolysis.

DelGEF, an RCC1-related protein encoded by a gene on chromosome 11p14 critical for two forms of hereditary deafness.

We have cloned a human cDNA, DELGEF ( afness ocus associated putative uanine nucleotide xchange actor), derived from a 225 kb genomic sequence of chromosome 11p14, critical for the Usher 1C syndrome and for DFNB18, a locus for non‐syndromic sensorineural deafness. The amino acid sequence of the protein hDelGEF1 is homologous to the nucleotide exchange factor RCC1 for the small GTPase Ran. hDelGEF2 is derived from the same DELGEF gene by alternative splicing. In addition, we have identified a murine homologue, mDelGEF. The ubiquitously expressed soluble protein hDelGEF1 is found both in the cytoplasm and in the nucleus. Overexpressed hDelGEF2 colocalizes with mitochondria.

Isolation of human NuMA protein

NuMA is a protein involved in maintenance of nuclear structure and in the assembly of the mitotic spindle. Expression of amino‐terminal deletion mutants results in a phenotype identical to that caused by a temperature‐sensitive defect of RCC1 (regulator of chromosome condensation). Here we describe the isolation of NuMA protein from HeLa cells under mild conditions as a prerequisite to study its interactions with elements of the RCC1‐Ran regulatory pathway. In an overlay assay, NuMA did not bind Ran·[γ‐32P]GTP. Thus it is clearly different from Ran·GTP binding proteins of similar Mr.