Activity-based protein profiling: The serine hydrolases

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors

We present evidence that the sporulation protein SpoIVFB of Bacillus subtilis is a member of a newly recognized family of metalloproteases that have catalytic centers adjacent to or within the membrane. SpoIVFB is required for converting the membrane-associated precursor protein, pro-σK, to the mature and active transcription factor σK by proteolytic removal of an N-terminal extension of 20 amino acids. SpoIVFB and other family members share the conserved sequence HEXXH, a hallmark of metalloproteases, as well as a second conserved motif NPDG, which is unique to the family. Both motifs, which are expected to form the catalytic center of the protease, overlap hydrophobic segments that are predicted to be separate transmembrane domains. The only other characterized member of this family of membrane-embedded metalloproteases is the mammalian Site-2 protease (S2P), which is required for the intramembrane cleavage of the eukaryotic transcription factor sterol regulatory element binding protein (SREBP). We report that amino acid substitutions in the two conserved motifs of SpoIVFB impair pro-σK processing and σK-directed gene expression during sporulation. These results and those from a similar analysis of S2P support the interpretation that both proteins are founding members of a family of metalloproteases involved in the activation of membrane-associated transcription factors. Thus, the pathways that govern the activation of the prokaryotic transcription factor pro-σK and the mammalian transcription factor SREBP not only are analogous but also use processing enzymes with strikingly homologous features.

Retinoic acid X receptor in the diploblast, Tripedalia cystophora

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (Kd = 4 × 10−10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His6)jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His6)jRXR showed that full-length jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.

Eukaryotic phytochromes: Light-regulated serine/threonine protein kinases with histidine kinase ancestry

The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes. Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.

Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

GCS1, an Arf Guanosine Triphosphatase-activating Protein in Saccharomyces cerevisiae, Is Required for Normal Actin Cytoskeletal Organization In Vivo and Stimulates Actin Polymerization In Vitro

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.