Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

Oligomeric structure of caveolin: implications for caveolae membrane organization.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.

The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal glycine of an invariably conserved GXXGXXK motif present in all steroid and phenol sulfotransferases for which primary structures are known. This consistency strongly suggests that all steroid and phenol sulfotransferase genes will be similarly spliced. The GXXGXXK motif forms the active binding site for the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate. Amino acid sequence alignment of 19 cloned steroid and phenol sulfotransferases starting with the GXXGXXK motif indicates that the 3'-terminal exon for each steroid and phenol sulfotransferase gene encodes a similarly sized C-terminal fragment of the protein. Interestingly, on further analysis of the alignment, three distinct amino acid sequence patterns emerge. The presence of the conserved functional GXXGXXK motif suggests that the protein domains encoded by steroid and phenol sulfotransferase 3'-terminal exons have evolved from a common ancestor. Furthermore, it is hypothesized that during the course of evolution, the 3'-terminal exon further diverged into at least three sulfotransferase subdivisions: a phenol or aryl group, an estrogen or phenolic steroid group, and a neutral steroid group.

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.

A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G alpha q and G alpha 11 proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio]triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G alpha q/G alpha 11 and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G alpha q/G alpha 11 and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G alpha q/G alpha 11 with actin. Large amounts of G alpha q/G alpha 11 were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G alpha q/G alpha 11 and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G alpha q/G alpha 11 proteins play a role in signal transduction.

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

Positive regulation of general transcription factor SIII by a tailed ubiquitin homolog.

General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.

Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.

Dissection of transcription factor TFIIF functional domains required for initiation and elongation.

TFIIF is unique among the general transcription factors because of its ability to control the activity of RNA polymerase II at both the initiation and elongation stages of transcription. Mammalian TFIIF, a heterodimer of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits, assists TFIIB in recruiting RNA polymerase II into the preinitiation complex and activates the overall rate of RNA chain elongation by suppressing transient pausing by polymerase at many sites on DNA templates. A major objective of efforts to understand how TFIIF regulates transcription has been to establish the relationship between its initiation and elongation activities. Here we establish this relationship by demonstrating that TFIIF transcriptional activities are mediated by separable functional domains. To accomplish this, we sought and identified distinct classes of RAP30 mutations that selectively block TFIIF activity in transcription initiation and elongation. We propose that (i) TFIIF initiation activity is mediated at least in part by RAP30 C-terminal sequences that include a cryptic DNA-binding domain similar to conserved region 4 of bacterial sigma factors and (ii) TFIIF elongation activity is mediated in part by RAP30 sequences located immediately upstream of the C terminus in a region proposed to bind RNA polymerase II and by additional sequences located in the RAP30 N terminus.

The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function.

In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N-terminal region with transcription factor IIB.

A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system.

We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.