SEARCH · PubMed Health

Results for “Multiprotein Complexes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,117 records · Page 62Linked to original sources

PubMed1997

MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains.

Membrane-associated guanylate kinase (MAGUK) proteins participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane at regions of cell-cell contact. MAGUKs are characterized by three types of protein-protein interaction modules: the PDZ domain, the Src homology 3 (SH3) domain, and the guanylate kinase (GuK) domain. The arrangement of these domains is conserved in all previously known MAGUKs: either one or three PDZ domains in the NH2-terminal half, followed by the SH3 domain, followed by a COOH-terminal GuK domain. In this report, we describe the cDNA cloning and subcellular distribution of MAGI-1, a MAGUK with three unique structural features: 1) the GuK domain is at the NH2 terminus, 2) the SH3 domain is replaced by two WW domains, and 3) it contains five PDZ domains. MAGI-1 mRNA was detected in several adult mouse tissues. Sequence analysis of overlapping cDNAs revealed the existence of three splice variants that are predicted to encode MAGI-1 proteins with different COOH termini. The longest variant, MAGI-1c, contains three bipartite nuclear localization signals in its unique COOH-terminal sequence and was found predominantly in the nucleus of Madin-Darby canine kidney cells. A shorter form lacking these signals was found primarily in membrane and cytoplasmic fractions. This distribution, which is reminiscent of that seen for the tight junction protein ZO-1, suggests that MAGI-1 may participate in the transmission of regulatory signals from the cell surface to the nucleus.

Amino Acid Sequence↗

PubMed1998

Progesterone regulates transcription of the p21(WAF1) cyclin- dependent kinase inhibitor gene through Sp1 and CBP/p300.

Progesterone has biphasic effects on proliferation of breast cancer cells; it stimulates growth in the first cell cycle, then arrests cells at G1/S of the second cycle accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21. We now show that progesterone regulates transcription of the p21 promoter by an unusual mechanism. This promoter lacks a canonical progesterone response element. Instead, progesterone receptors (PRs) interact with the promoter through the transcription factor Sp1 at the third and fourth of six Sp1 binding sites located downstream of nucleotide 154. Mutation of Sp1 site 3 eliminates basal transcription, and mutation of sites 3 and 4 eliminates transcriptional up-regulation by progesterone. Progesterone-mediated transcription is further prevented by overexpression of E1A, suggesting that CBP/p300 is required. Indeed, in HeLa cells, Sp1 and CBP/p300 associate with stably integrated flag-tagged PRs in a multiprotein complex. Since many signals converge on p21, cross-talk between PRs and other factors co-localized on the p21 promoter, may explain how progesterone can be either proliferative or differentiative in different target cells.

Cyclin-Dependent Kinase Inhibitor p21↗

PubMed1998

Plasma membrane Ca2+ ATPase isoform 4b binds to membrane-associated guanylate kinase (MAGUK) proteins via their PDZ (PSD-95/Dlg/ZO-1) domains.

Plasma membrane Ca2+ ATPases are P-type pumps important for intracellular Ca2+ homeostasis. The extreme C termini of alternatively spliced "b"-type Ca2+ pump isoforms resemble those of K+ channels and N-methyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains. Yeast two-hybrid assays demonstrated strong interaction of Ca2+ pump 4b with the PDZ1 + 2 domains of several mammalian MAGUKs. Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins. Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1 + 2 domains of hDlg with nanomolar affinity (KD = 1.6 nM), whereas binding to PDZ3 was in the micromolar range (KD = 1.2 microM). In contrast, the corresponding C-terminal peptide of Ca2+ pump 2b interacted weakly with PDZ1 + 2 and not at all with PDZ3 of hDlg. Ca2+ pump 4b bound strongly to PDZ1 + 2 + 3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind. Together, these data demonstrate a direct physical binding of Ca2+ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca2+ pumps. Alternative splicing may dictate their specific interaction with PDZ domain-containing proteins, potentially influencing their localization and incorporation into functional multiprotein complexes at the plasma membrane.

Alternative Splicing↗

PubMed1998

Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

Animals↗

PubMed1998

Identification of highly conserved amino-terminal segments of dTAFII230 and yTAFII145 that are functionally interchangeable for inhibiting TBP-DNA interactions in vitro and in promoting yeast cell growth in vivo.

TFIID is a multiprotein complex composed of TBP and several TAFIIs. Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAFII230 (dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAFII145 TAND (yTAND) was divided into two subdomains, yTANDI10-37 and yTANDII46-71, that function cooperatively. Here, we identify dTANDII within the amino terminus of dTAFII230 at 118-143 amino acids in addition to dTANDI18-77, reported previously. dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 degreesC. In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.

Amino Acid Sequence↗

PubMed1998

Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein.

Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.

Amino Acid Sequence↗

PubMed1999

Identification of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated kinase-PP2A.

A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A's important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258-1261) and casein kinase IIalpha (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.

Amino Acid Sequence↗

PubMed1999

Exo84p is an exocyst protein essential for secretion.

The exocyst is a multiprotein complex that plays an important role in secretory vesicle targeting and docking at the plasma membrane. Here we report the identification and characterization of a new component of the exocyst, Exo84p, in the yeast Saccharomyces cerevisiae. Yeast cells depleted of Exo84p cannot survive. These cells are defective in invertase secretion and accumulate vesicles similar to those in the late sec mutants. Exo84p co-immunoprecipitates with the exocyst components, and a portion of the Exo84p co-sediments with the exocyst complex in velocity gradients. The assembly of Exo84p into the exocyst complex requires two other subunits, Sec5p and Sec10p. Exo84p interacts with both Sec5p and Sec10p in a two-hybrid assay. Overexpression of Exo84p selectively suppresses the temperature sensitivity of a sec5 mutant. Exo84p specifically localizes to the bud tip or mother/daughter connection, sites of polarized secretion in the yeast S. cerevisiae. Exo84p is mislocalized in a sec5 mutant. These studies suggest that Exo84p is an essential protein that plays an important role in polarized secretion.

Amino Acid Sequence↗

PubMed1999

The role of FAST-1 and Smads in transcriptional regulation by activin during early Xenopus embryogenesis.

Smads are signal transducers for the transforming growth factor-beta superfamily of factors. In early Xenopus embryos, the transforming growth factor-beta member activin induces the gene Mix.2 by stimulating the formation of a multiprotein complex, activin-responsive factor (ARF). This complex contains Smad2 or Smad3, Smad4, and a novel forkhead transcription factor, FAST-1, and binds to an enhancer (activin-responsive element; ARE) that confers activin regulation of Mix.2 transcription. Both FAST-1 and Smads can bind directly to the ARE; we have investigated 1) the role of FAST-1 and Smad DNA binding sites in ARF recognition of the ARE, 2) the contributions of FAST-1 and Smad binding to ARF binding in vitro and to ARE regulation in early Xenopus embryos, 3) the extent to which different Smads can replace Smad4 in regulation of the ARE. We find that ARF binds to ARE through both FAST-1 and Smad binding sites. FAST-1 recognition of the ARE is essential both for ARF binding in vitro and activin regulation in vivo. In contrast, Smad binding of ARE is unnecessary for ARF binding or activin regulation but does enhance the binding and regulatory activity of ARF. Also, Smad3 can partially substitute for Smad4 in the regulation of the ARE. These observations elucidate how broadly expressed signal transducers (Smads) regulate a developmentally specific transcriptional response in conjunction with a temporally restricted transcription factor, FAST-1.

Activins↗

PubMed1999

Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries.

Transcription factor IIIA (TFIIIA) binds to the 5 S rRNA gene through its zinc finger domain and directs the assembly of a multiprotein complex that promotes transcription initiation by RNA polymerase III. Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DNA binding and in vitro transcription analyses, have been used herein to investigate the domain organization and zinc requirements of Saccharomyces cerevisiae TFIIIA. Species containing either nine, six, or three zinc equivalents were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA. Partial digestion of the metal-saturated, 9 Zn2+-liganded factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a C-terminal portion including the ninth finger. Proteolyzed TFIIIA has the same 5 S DNA binding ability of the intact protein yet no longer supports in vitro 5 S rRNA synthesis. Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only containing 3 zinc ions are severely compromised. In contrast, the 6 Zn2+-liganded species was found to be indistinguishable from metal-saturated TFIIIA. By demonstrating the existence of three classes of zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexibility built into this complex transcription factor.

Binding Sites↗

PubMed1999

Genetic dissection of hTAF(II)130 defines a hydrophobic surface required for interaction with glutamine-rich activators.

The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAF(II)s). The central domain of human TAF(II)130 contains four glutamine-rich regions Q1-Q4 that interact with transcriptional activators such as Sp1 and CREB and mediate activation. We screened in yeast random point mutations introduced into Q1-Q4 against the Sp1 activation domain and obtained a distinct set of hTAF(II)130s with alterations in TAF(II)-activator interaction. Here we characterize functionally an hTAF(II)130 mutant containing a phenylalanine to serine change at position 311 (F311S) that is compromised in its ability to associate with Sp1B and CREB-N activation domains. Substitution of phenylalanine with tyrosine but not with isoleucine or tryptophan also reduced hTAF(II)130 interaction, suggesting that the hydrophobic character rather than the specific amino acid at this position is a key determinant of interaction. Deletion of nine amino acids (Delta9) surrounding Phe(311) abolished the interaction of hTAF(II)130 with Sp1. Overexpression of hTAF(II)130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Delta9 only partially suppressed Sp1-mediated activation. Thus, a short hydrophobic sequence motif encompassing Phe(311) in hTAF(II)130 represents a critical surface with which Sp1B interacts to activate transcription.

Amino Acid Sequence↗

PubMed1999

The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2.

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.

Amino Acid Substitution↗

PubMed2000

Human homologue of the Drosophila discs large tumor suppressor protein forms an oligomer in solution. Identification of the self-association site.

The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superfamily, interacts with K(+) channels, N-methyl-d-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains. MAGUKs play pivotal roles in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses. To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells. Gel permeation chromatography of full-length hDlg revealed that the purified protein migrates as a large particle of size >440 kDa. Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal domain. Ultrastructural analysis of hDlg by low angle rotary shadow electron microscopy revealed that the full-length hDlg protein as well as its amino-terminal domain exhibits a highly flexible irregular shape. Further evaluation of the self-association state of hDlg using sedimentation equilibrium centrifugation, matrix-assisted laser desorption/ionization mass spectrometry, and chemical cross-linking techniques confirmed that the oligomerization site of hDlg is contained within its amino-terminal domain. This unique amino-terminal domain mediates multimerization of hDlg into dimeric and tetrameric species in solution. Sedimentation velocity experiments demonstrated that the oligomerization domain exists as an elongated tetramer in solution. In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self-association. Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K(+) channels and dimeric adenomatous polyposis coli tumor suppressor protein. Our findings, therefore, begin to rationalize the role of hDlg in the clustering of membrane channels and formation of multiprotein complexes necessary for signaling and cell proliferation pathways.

Animals↗

PubMed2000

Deletion of PBP/PPARBP, the gene for nuclear receptor coactivator peroxisome proliferator-activated receptor-binding protein, results in embryonic lethality.

We previously isolated and identified peroxisome proliferator-activated receptor (PPAR)-binding Protein (PBP) as a coactivator for PPARgamma. PBP has recently been identified as a component of the multiprotein complexes such as TRAP, DRIP, and ARC that appear to play an important role in the transcriptional activation by several transcriptional factors including nuclear receptors. To assess the biological significance of PBP, we disrupted the PBP gene (PBP/PPARBP) in mice by homologous recombination. PBP(+/-) mice are healthy, fertile, and do not differ significantly from PBP(+/+) control littermates. PBP null mutation (PBP(-/-)) is embryonically lethal at embryonic day 11.5, suggesting that PBP is an essential gene for mouse embryogenesis. The embryonic lethality is attributed, in part, to defects in the development of placental vasculature similar to those encountered in PPARgamma mutants. Transient transfection assays using fibroblasts isolated from PBP mutant embryos revealed a decreased capacity for ligand-dependent transcriptional activation of PPARgamma as compared with fibroblasts derived form the wild type embryos. These observations suggest that there is no functional redundancy between PBP and other coactivators such as steroid receptor coactivator-1 and that PBP plays a critical role in the signaling of PPARgamma and other nuclear receptors.

Animals↗

PubMed2002

COMPASS, a histone H3 (Lysine 4) methyltransferase required for telomeric silencing of gene expression.

The trithorax (Trx) family of proteins is required for maintaining a specific pattern of gene expression in some organisms. Recently we reported the isolation and characterization of COMPASS, a multiprotein complex that includes the Trx-related protein Set1 of the yeast Saccharomyces cerevisiae. Here we report that COMPASS catalyzes methylation of the fourth lysine of histone H3 in vitro. Set1 and several other components of COMPASS are also required for histone H3 methylation in vivo and for transcriptional silencing of a gene located near a chromosome telomere.

Chromosomes, Fungal↗

PubMed2000

hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions.

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.

Binding Sites↗

PubMed2000

The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and -2.

Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice.

Adaptor Proteins, Signal Transducing↗

PubMed2000

HEAT repeats mediate plasma membrane localization of Tor2p in yeast.

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.

Amino Acid Sequence↗