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M B Yaffe

Publications and source records attributed to M B Yaffe.

45 records · Page 3Linked to original sources
PubMed1992

Characterization of the adenovirus E3 protein that down-regulates the epidermal growth factor receptor. Evidence for intermolecular disulfide bonding and plasma membrane localization.

We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tollefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S. M. (1989) Cell 57, 135-144). Previous studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10, 5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S., Carlin, C. R., and Wold, W. S. M. (1990) J. Virol. 64, 794-801). We report here that the 13.7- and 11.3-kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with 125I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radiolabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor.

Adenovirus Early Proteins↗
PubMed1992

Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.

Involucrin is a 68-kDa precursor of the keratinocyte cornified envelope. During keratinocyte terminal differentiation glutamine residues of involucrin become covalently cross-linked to other envelope precursors via covalent epsilon-(gamma-glutamyl)lysine bonds. In the present study we examine the secondary and tertiary structure of human involucrin using computer algorithms, circular dichroism, and electron microscopy. Our results indicate that involucrin is an extended, flexible, rod-shaped molecule that has a length of 460 A, an axial ratio of 30:1 and possesses between 50 and 75% alpha-helical content. Glutamine residues are circumferentially distributed along the length of the alpha-helical segments of the molecule, a distribution that is conserved in all species. We hypothesize that this distribution of glutamine residues together with the elongated shape of the molecule permits optimal interaction of involucrin glutamyl side chains with the lysine residues of other para-membranous proteins during transglutaminase-mediated cross-linking. Moreover, its long length allows involucrin to cross-link molecules that are separated by substantial distances in the cornified envelope. These properties allow a single involucrin molecule to form multiple cross-links, in multiple spatial planes, with other envelope precursors. Thus, the structure of involucrin is that of an ideal intermolecular cross-bridge.

Amino Acid Sequence↗
PubMed1990

Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein.

Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit.

Animals↗
PubMed1989

Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers.

Cell-free translation of beta-tubulin mRNA generates full length beta-tubulin polypeptides distributed in three molecular forms: a high molecular weight lysate-associated form, the free beta-tubulin subunit, and the alpha beta-heterodimer (Yaffe, M.B., Farr, G. W., and Sternlicht, H. (1988) J. Biol. Chem. 263, 16023-16031). A quantitative assay system for these three forms was developed and used to measure the rates of incorporation/exchange of the newly synthesized free beta-subunit and the high molecular weight form into tubulin heterodimers following incubation of the 35S-translation products with unlabeled bovine tubulin dimer. This exchange process was found to be slow and strongly temperature-dependent. The half-lives for exchange ranged from 12.5 min at 37 degrees C to 17.5 h at 0 degree C with a measured activation energy of 22.5 kcal/mol. Microtubule-associated proteins appeared to play no role in the exchange process, since identical exchange rates were observed regardless of whether microtubule protein or phosphocellulose-purified tubulin was used as the source of tubulin dimer. Surprisingly, the exchange rates were found to be independent of dimer concentration. We interpret these results as evidence for a rate-limiting, slow conformational change that occurs within the newly synthesized beta-subunits prior to their association with alpha-tubulin to generate the alpha beta-hetero-dimer.

Animals↗
PubMed1988

Translation of beta-tubulin mRNA in vitro generates multiple molecular forms.

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.

Animals↗
PubMed1988

Expression of a human alpha-tubulin: properties of the isolated subunit.

We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.

Alkaloids↗
PubMed1987

A model of the nucleotide-binding site in tubulin.

Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.

Amino Acid Sequence↗
PubMed1986

Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin.

Previous studies have shown that tubulin, a major protein component of the microtubule, is rendered assembly incompetent when a highly reactive lysine residue (HRL) in the alpha polypeptide of tubulin dimer is reductively methylated [cf. Sherman, G., Rosenberry, T. L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156]. In this study we demonstrate that the HRL in bovine brain tubulin is Lys-394, a residue proximal in the alpha-tubulin sequence to the highly negatively charged carboxy-terminus region (residues 412-450) previously implicated in assembly. pH studies were undertaken to probe the local environment of Lys-394. These studies indicated that Lys-394 reactivity toward HCHO is sensitive to the titration of a pKa 6.3 group presumed to be a histidine residue. This assignment is supported by our finding that histidine modification via diethyl pyrocarbonate strongly affects Lys-394 reactivity toward HCHO as well as microtubule assembly. We propose on the basis of secondary structure considerations and published sequence data for a variety of tubulins that Lys-394 is part of an evolutionarily conserved cluster of basic residues (effective charge: 2+ to 2.5+ at neutral pH) composed of Lys-394, His-393, and Arg-390, which is important for tubulin function and which renders Lys-394 reactive as a nucleophile.

Amino Acid Sequence↗