Nuclear Domains Enriched in RNA 3′-processing Factors Associate with Coiled Bodies and Histone Genes in a Cell Cycle–dependent Manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3′-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle–dependent expression of histone genes.

Pex17p Is Required for Import of Both Peroxisome Membrane and Lumenal Proteins and Interacts with Pex19p and the Peroxisome Targeting Signal–Receptor Docking Complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Δ mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Δ mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal–receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by Site-2 protease

The NH2-terminal domains of membrane-bound sterol regulatory element-binding proteins (SREBPs) are released into the cytosol by regulated intramembrane proteolysis, after which they enter the nucleus to activate genes encoding lipid biosynthetic enzymes. Intramembrane proteolysis is catalyzed by Site-2 protease (S2P), a hydrophobic zinc metalloprotease that cleaves SREBPs at a membrane-embedded leucine-cysteine bond. In the current study, we use domain-swapping methods to localize the residues within the SREBP-2 membrane-spanning segment that are required for cleavage by S2P. The studies reveal a requirement for an asparagine-proline sequence in the middle third of the transmembrane segment. We propose a model in which the asparagine-proline sequence serves as an NH2-terminal cap for a portion of the transmembrane α-helix of SREBP, allowing the remainder of the α-helix to unwind partially to expose the peptide bond for cleavage by S2P.

Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)–phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1–Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392–541) directly interacts with the NH2-terminal domain of Stat1 (amino acids 1–191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH2-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1–Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1.

The fourth chromosome of Drosophila melanogaster: Interspersed euchromatic and heterochromatic domains

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50–75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708–831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na+- and K+-dependent pH homeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H+, Na+/H+, and K+/H+ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H+ and Na+/H+ antiports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

Determinant for β-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus

The pore-forming α subunit of large conductance voltage- and Ca2+-sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers β-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 Å resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a “minichaperone.” This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

Domains of P2X receptors involved in desensitization

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X1 (or P2X3) receptors had strong desensitization, whereas currents in cells expressing P2X2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X1 receptor of either one of two 34-residue segments from the P2X2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X2 receptor only by providing both these segments of the P2X1 (or P2X3) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

Staphylococcal protein A: Unfolding pathways, unfolded states, and differences between the B and E domains

We present multiple native and denaturation simulations of the B and E domains of the three-helix bundle protein A, totaling 60 ns. The C-terminal helix (H3) consistently denatures later than either of the other two helices and contains residual helical structure in the denatured state. These results are consistent with experiments suggesting that the isolated H3 fragment is more stable than H1 and H2 and that H3 forms early in folding. Interestingly, the denatured state of the B domain is much more compact than that of the E domain. This sequence-dependent effect on the dimensions of the denatured state and the lack of correlation with structure suggest that the radius of gyration can be a misleading reaction coordinate for unfolding/folding. Various unfolding and refolding events are observed in the denaturation simulations. In some cases, the transitions are facilitated through interactions with other portions of the protein—contact-assisted helix formation. In the native simulations, the E domain is very stable: after 6 ns, the Cα root-mean-square deviation from the starting structure is less than 1.4 Å. In contrast, the native state of the B domain deviates more and its inter-helical angles fluctuate. In apparent contrast, we note that the B domain is thermodynamically more stable than the E domain. The simulations suggest that the increased stability of the B domain may be due to heightened mobility, and therefore entropy, in the native state and decreased mobility/entropy in the more compact denatured state.

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to Gβ5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gβ subunits, only RGS6 and Gβ5 interact. The expression of mRNA for RGS6 and Gβ5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of Gβ binding by GGL domain mutants, support the contention that Gγ-like regions within RGS proteins interact with Gβ5 subunits in a fashion comparable to conventional Gβ/Gγ pairings. Mutation of the highly conserved Phe-61 residue of Gγ2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gβ5/Gγ2 heterodimer, highlighting the importance of this residue to GGL/Gβ5 association.