Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140

We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor-alpha (ER) to region E in vivo. In this system, the cDNAs corresponding to the A-D, E, E/F, A-E (deltaF), and full-length (wtER) domains of the human ER were each cloned into the yeast two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations in yeast harboring a GAL1-lacZ reporter. The reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity. We found that the interaction of E or E/F domains of the ER with full-length ER is estradiol dependent and estrogen responsive element independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions. In the presence of F domain, this activity is reduced 10-fold. The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region. We propose that the full-length ER contains intrinsic dimerization restraints contributed by regions outside domain E that are released upon binding hormone agonist. In addition, we have demonstrated that coactivator RIP140 is able to interact with the ER in vivo at the E domain of the receptor in the presence of estrogen. Yeast two-hybrid analysis shows that RIP140 does not homodimerize in the presence or absence of estrogens. We present evidence showing that the ER has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the ER itself that are present outside of the E domain compromise this ability.     

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.     

Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.     

Evidence for PDZ domains in bacteria, yeast, and plants

Several dozen signaling proteins are now known to contain 80-100 residue repeats, called PDZ (or DHR or GLGF) domains, several of which interact with the C-terminal tetrapeptide motifs X-Ser/Thr-X-Val-COO- of ion channels and/or receptors. PDZ domains have previously been noted only in mammals, flies, and worms, suggesting that the primordial PDZ domain arose relatively late in eukaryotic evolution. Here, techniques of sequence analysis-including local alignment, profile, and motif database searches-indicate that PDZ domain homologues are present in yeast, plants, and bacteria. It is suggested that two PDZ domains occur in bacterial high-temperature requirement A (htrA) and one in tail-specific protease (tsp) homologues, and that a yeast htrA homologue contains four PDZ domains. Sequence comparisons suggest that the spread of PDZ domains in these diverse organisms may have occurred via horizontal gene transfer. The known affinity of Escherichia coli tsp for C-terminal polypeptides is proposed to be mediated by its PDZ-like domain, in a similar manner to the binding of C-terminal polypeptides by animal PDZ domains.     

Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding

The yeast adenylyl cyclase-associated protein, CAP, was identified as a component of the RAS-activated cyclase complex. CAP consists of two functional domains separated by a proline-rich region. One domain, which localizes to the amino terminus, mediates RAS signaling through adenylyl cyclase, while a domain at the carboxyl terminus is involved in the regulation of cell growth and morphogenesis. Recently, the carboxyl terminus of yeast CAP was shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear. Here, we demonstrate that the carboxyl-terminal domains of CAP and CAP homologs have two separate functions. We show that carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin. We also show that this domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers. The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding. Finally, we present evidence that links a segment of the proline-rich region of CAP to its localization in yeast. Together, these results suggest that all three domains of CAP proteins are functional.     

Inhibition of the interaction between tyrosine-based motifs and the medium chain subunit of the AP-2 adaptor complex by specific tyrphostins

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs found within the cytosolic domains of certain integral membrane proteins. Many of these tyrosine motifs conform to the consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi motif has been shown to interact with the medium chain subunits of adaptor complexes that generally link relevant integral membrane protein cytosolic domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is also very similar to motifs that are targets for phosphorylation by tyrosine kinases. Tyrosine kinase inhibitors known as tyrphostins are structural analogues of tyrosine, and so it is possible that tyrphostins could also inhibit interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain. We have previously shown that this motif interacts directly with the medium chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We have investigated a range of tyrphostins and demonstrated a specific inhibition of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23 through in vitro analysis and the yeast two-hybrid system. These data raise the exciting possibility that different membrane traffic events could be inhibited by specific tyrphostins.     

Essential binding and functional domains of human bleomycin hydrolase

Bleomycin hydrolase (BH) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin, and contains a unique C-terminal amino acid sequence. We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay. To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH1-82) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH14-103) and another neighboring the C-terminus (hBH358-455). In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted. The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation. Thus, the C-terminus of hBH has two independent roles controlling both the catalytic activity and oligomerization of hBH.     

Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain

The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains. We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome. The approximately 2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel approximately 84-kDa Golgi protein we termed golgin-84. Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus. In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an approximately 400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-II oncogene. Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.     

The second catalytic domain of protein tyrosine phosphatase delta (PTP delta) binds to and inhibits the first catalytic domain of PTP sigma

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTP delta, and PTP sigma, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTP sigma (PTP sigma-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTP delta (PTP delta-D2) as an interactor of PTP sigma-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTP sigma-D1 and PTP delta-D2, an association which required the presence of the wedge sequence in PTP sigma-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTP alpha. This interaction was not reciprocal, as PTP delta-D1 did not bind PTP sigma-D2. Addition of a glutathione S-transferase (GST)-PTP delta-D2 fusion protein (but not GST alone) to GST-PTP sigma-D1 led to approximately 50% inhibition of the catalytic activity of PTP sigma-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTP sigma-D1 activity was obtained with coimmunoprecipitated PTP delta-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTP sigma (PTP sigma-D2), very similar in sequence to PTP delta-D2, bound poorly to PTP sigma-D1. PTP delta-D1 and LAR-D1 were also able to bind PTP delta-D2, but more weakly than PTP sigma-D1, with a binding hierarchy of PTP sigma-D1 > PTP delta-D1 > LAR-D1. These results suggest that association between PTP sigma-D1 and PTP delta-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.     

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1

The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.