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H G Dohlman

Publications and source records attributed to H G Dohlman.

At least 19 recordsLinked to original sources
PubMed2001

Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast.

Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome. Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo. As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety. Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12. Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7. These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK.

Amino Acid Sequence↗
PubMed2001

Regulation of G protein-initiated signal transduction in yeast: paradigms and principles.

All cells have the capacity to evoke appropriate and measured responses to signal molecules (such as peptide hormones), environmental changes, and other external stimuli. Tremendous progress has been made in identifying the proteins that mediate cellular response to such signals and in elucidating how events at the cell surface are linked to subsequent biochemical changes in the cytoplasm and nucleus. An emerging area of investigation concerns how signaling components are assembled and regulated (both spatially and temporally), so as to control properly the specificity and intensity of a given signaling pathway. A related question under intensive study is how the action of an individual signaling pathway is integrated with (or insulated from) other pathways to constitute larger networks that control overall cell behavior appropriately. This review describes the signal transduction pathway used by budding yeast (Saccharomyces cerevisiae) to respond to its peptide mating pheromones. This pathway is comprised by receptors, a heterotrimeric G protein, and a protein kinase cascade all remarkably similar to counterparts in multicellular organisms. The primary focus of this review, however, is recent advances that have been made, using primarily genetic methods, in identifying molecules responsible for regulation of the action of the components of this signaling pathway. Just as many of the constituent proteins of this pathway and their interrelationships were first identified in yeast, the functions of some of these regulators have clearly been conserved in metazoans, and others will likely serve as additional models for molecules that carry out analogous roles in higher organisms.

Fungal Proteins↗
PubMed2000

Endoproteolytic processing of Sst2, a multidomain regulator of G protein signaling in yeast.

Regulators of G protein signaling (RGS proteins) constitute a large family of G protein-binding proteins. All RGS proteins contain a conserved core domain that can accelerate G protein GTPase activity. In addition, many family members contain a unique N-terminal domain of unknown function. Here, we demonstrate that the RGS protein in yeast, Sst2, is proteolytically processed in vivo to yield separate but functional N-terminal and RGS core domain fragments. In whole cell lysates, the full-length SST2 product (82 kDa) as well as a prominent 36-kDa species are specifically recognized by antibodies against the C terminus of the Sst2 protein. Purification and chemical sequencing of the 36-kDa species revealed cleavage sites after Ser-414 and Ser-416, just preceding the region of RGS homology. Expression of a mutationally truncated form of the protein (C-Sst2) could not restore function to an sst2Delta mutant strain. In contrast, co-expression of C-Sst2 with the N-terminal domain (N-Sst2) partially restored the ability to regulate the growth arrest response but not the transcription induction response. Whereas the full-length protein was localized to the microsomal and plasma membrane fractions, the N-Sst2 species was predominantly in the microsomal fraction, and C-Sst2 was in the soluble fraction. Mutations that block proteasome or vacuolar protease function, or mutations in the cleavage site Ser residues of Sst2, did not alter processing. However, Sst2 processing did require expression of other components of the pheromone response pathway, including the receptor and the G protein. These results indicate that Sst2 is proteolytically processed, that this event is regulated by the signaling pathway, and that processing can profoundly alter the function and subcellular localization of the protein.

Amino Acid Sequence↗
PubMed2000

Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast.

Mammalian phosducins are known to bind G protein betagamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gbetagamma. Binding was enhanced by pheromone stimulation and by the addition of GTPgammaS, conditions that favor dissociation of Gbetagamma from Galpha. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast.

The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.

Apoptosis↗
PubMed2000

Use of G-protein fusions to monitor integral membrane protein-protein interactions in yeast.

The control of protein-protein interactions is a fundamental aspect of cell regulation. Here we describe a new approach to detect the interaction of two proteins in vivo. By this method, one binding partner is an integral membrane protein whereas the other is soluble but fused to a G-protein gamma-subunit. If the binding partners interact, G-protein signaling is disrupted. We demonstrate interaction between known binding partners, syntaxin 1a with neuronal Sec1 (nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1. In addition, we describe a genetic screen to identify nSec1 mutants that are expressed normally, but are no longer able to bind to syntaxin 1a. This provides a convenient method to study interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Feedback phosphorylation of an RGS protein by MAP kinase in yeast.

Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling.

Amino Acid Sequence↗
PubMed1998

Second site suppressor mutations of a GTPase-deficient G-protein alpha-subunit. Selective inhibition of Gbeta gamma-mediated signaling.

G proteins transmit signals from cell surface receptors to intracellular effectors. The intensity of the signal is governed by the rates of GTP binding (leading to subunit dissociation) and hydrolysis. Mutants that cannot hydrolyze GTP (e.g. GsalphaQ227L, Gi2alphaQ205L) are constitutively activated and can lead to cell transformation and cancer. Here we have used a genetic screen to identify intragenic suppressors of a GTPase-deficient form of the Galpha in yeast, Gpa1(Q323L). Sequencing revealed second-site mutations in three conserved residues, K54E, R327S, and L353Delta (codon deletion). Each mutation alone results in a complete loss of the beta gamma-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian Gi2alpha (K46E, R209S, L235Delta) lead to inhibition of Gbeta gamma-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these beta gamma inhibitors (R209S) has no effect on Gi2alpha-mediated regulation of adenylyl cyclase. Despite its impaired ability to release beta gamma, purified recombinant Gpa1(R327S) is fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling Gbeta gamma- and Galpha-mediated signaling events in whole cells and animals. In addition, they serve as a model for drugs that could directly inhibit G protein activity and cell transformation.

Animals↗
PubMed1998

A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins.

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Enzyme Activation↗
PubMed1998

Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast.

Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo. Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro. A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone. Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchange chromatography. Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations. These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.

Escherichia coli↗
PubMed1998

Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit.

Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors that propagate a signal. G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G protein alpha-subunits in vitro. It is not known, however, if Galpha GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a Galpha mutant in yeast (gpa1(sst)) that phenotypically mimics the loss of its cognate RGS protein (SST2). The gpa1(sst) mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activity in vitro. The analogous mutation in a mammalian Gqalpha is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through Galpha and that RGS-GAP activity is responsible for their desensitizing activity in cells. The Galphasst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.

Animals↗
PubMed1998

Regulation of G protein signalling in yeast.

A common property of cell signaling systems is the ability to adapt to chronic stimulation. A genetic analysis of receptor/G protein signaling in yeast has led to the identification of a new class of regulators of G protein signaling (RGS proteins), as well as to new insights about the regulatory role of G protein modifications (myristoylation, palmitoylation). Similar modes of regulation are now known to exist in humans. These discoveries fill some important gaps in our understanding of signal transduction, and provide an instructive example of how model organisms, like yeast, can provide new insights relevant to signal regulation in higher eukaryotes.

Animals↗
PubMed1998

N-terminal domain of Gpa1 (G protein alpha) subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae.

G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein alpha subunit are necessary for membrane attachment. Here we identify a region within the yeast G alpha (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsalpha with the corresponding sequence from Gpa1. Gsalpha is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsaplha chimeras were assayed for proper membrane localization by functional complementation of a gpa1Delta ;) mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.

Amino Acid Sequence↗
PubMed1997

Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain.

The recently discovered regulators of G-protein signaling (RGS) proteins potently modulate the functioning of heterotrimeric G-proteins by stimulating the GTPase activity of G-protein alpha subunits. The mRNAs for numerous subtypes of putative RGS proteins have been identified in mammalian tissues, but little is known about their expression in brain. We performed a systematic survey of the localization of mRNAs encoding nine of these RGSs, RGS3-RGS11, in brain by in situ hybridization. Striking region-specific patterns of expression were observed. Five subtypes, RGS4, RGS7, RGS8, RGS9, and RGS10 mRNAs, are densely expressed in brain, whereas the other subtypes (RGS3, RGS5, RGS6, and RGS11) are expressed at lower density and in more restricted regions. RGS4 mRNA is notable for its dense expression in neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus. RGS8 mRNA is highly expressed in the cerebellar Purkinje cell layer as well as in several midbrain nuclei. RGS9 mRNA is remarkable for its nearly exclusive enrichment in striatal regions. RGS10 mRNA is densely expressed in dentate gyrus granule cells, superficial layers of neocortex, and dorsal raphe. To assess whether the expression of RGS mRNAs can be regulated, we examined the effect of an acute seizure on levels of RGS7, RGS8, and RGS10 mRNAs in hippocampus. Of the three subtypes, changes in RGS10 levels were most pronounced, decreasing by approximately 40% in a time-dependent manner in response to a single seizure. These results, which document highly specific patterns of RGS mRNA expression in brain and their ability to be regulated in a dynamic manner, support the view that RGS proteins may play an important role in determining the intensity and specificity of signaling pathways in brain as well as their adaptations to synaptic activity.

Animals↗
PubMed1996

Identification of Triton X-100 insoluble membrane domains in the yeast Saccharomyces cerevisiae. Lipid requirements for targeting of heterotrimeric G-protein subunits.

Low density Triton X-100 insoluble (LDTI) membrane domains are found in most mammalian cell types. Previous biochemical and immunolocalization studies have revealed the presence of G-protein coupled receptors and heterotrimeric G-protein subunits (Galpha and Gbetagamma subunits) within these structures, implicating mammalian LDTI membrane domains in G-protein coupled signaling. Here, we present biochemical evidence that similar LDTI structures exist in a genetically tractable organism, the yeast Saccharomyces cerevisiae. Yeast LDTI membranes were purified based on the known biochemical properties of mammalian LDTI membranes: (i) their Triton X-100 insolubility; and (ii) their discrete buoyant density in sucrose gradients. As with purified mammalian LDTI membranes, these yeast LDTI membranes harbor the subunits of the heterotrimeric G-proteins (Galpha and Gbetagamma subunits). Other plasma membrane marker proteins (the plasma membrane H+-ATPase and a GPI-linked protein Gas1p) are preferentially excluded from these purified fractions. Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast membrane domains. We find that the targeting of Galpha is independent of myristoylation, whereas targeting of Ggamma requires prenylation. Perhaps surprisingly, the targeting of Gbeta to this membrane domain did not require coexpression of Ggamma. It should now be possible to dissect the function of LDTI membrane domains using yeast as a model genetic system.

Cell Compartmentation↗
PubMed1996

Partial constitutive activation of pheromone responses by a palmitoylation-site mutant of a G protein alpha subunit in yeast.

G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus. The G protein alpha subunit in the yeast Saccharomyces cerevisiae (GPA1 gene product, Gpa1p) is known to be myristoylated, and this modification is essential for G protein activity in vivo. Here we examined whether Gpa1p is palmitoylated and determined the functional consequences of this modification. [3H]-Palmitic acid was incorporated into Gpa1p in cells expressing myc-tagged Gpa1p or Gpa1p-Gst. The label was released upon hydroxylamine treatment. Substitution of the conserved Cys 3 for Ser blocked incorporation of the label (Gpa1pC3S). Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation had no effect on myristoylation of Gpa1p. Gpa1pC3S complemented the gpa1 delta mutation in vivo and formed a complex with G beta gamma that was able to undergo nucleotide exchange in vitro. However, basal and pheromone-induced FUSl-lacZ transcription were 2-5-fold higher in the C3S mutant. Pheromone-induced growth arrest was also enhanced by the mutation, but recovery from arrest was not affected. Like wild-type Gpa1p, the C3S mutant was predominantly membrane-associated. Upon Triton X-114 partitioning or high pH treatment, no difference in the membrane-binding properties of the wild-type Gpa1p and the C3S mutant was detected. By sucrose density gradient centrifugation of membranes, however, most of the mutant protein was mislocalized to a non-plasma membrane compartment, whereas G beta gamma localization was unaltered. Taken together, our data suggest that Gpa1p is palmitoylated via a thioester bond at Cys 3 and that palmitoylation plays a role in modulating Gpa1p signaling and membrane localization.

Cell Membrane↗
PubMed1996

Regulation of membrane and subunit interactions by N-myristoylation of a G protein alpha subunit in yeast.

Initiation of the mating process in yeast Saccharomyces cerevisiae requires the action of secreted pheromones and G protein-coupled receptors. As in other eukaryotes, the yeast G protein alpha subunit undergoes N-myristoylation (GPA1 gene product, Gpa1p). This modification appears to be essential for function, since a myristoylation site mutation exhibits the null phenotype in vivo (gpa1(G2A)). Here we examine how myristoylation affects Gpa1p activity in vitro. We show that the G2A mutant of Gpa1p, when fused with glutathione S-transferase, can still form a complex with the G protein betagamma subunits. The complex is stabilized by GDP and is dissociated upon treatment with guanosine 5'-O-(thiotriphosphate). In addition, there is no apparent difference in the relative binding affinity of Gbetagamma for mutant and wild-type Gpa1p. Using sucrose density gradient fractionation of cell membranes, Gpa1p associates normally with the plasma membrane whereas Gpa1pG2A is mislocalized to a microsomal membrane fraction. A portion of Gbetagamma is also mislocalized in these cells, as it is in a gpa1Delta strain. In contrast, wild-type Gpa1p reaches the plasma membrane in cells that do not express Gbetagamma or cell surface receptors. These findings indicate that mislocalization of Gpa1pG2A is not caused by a redistribution of Gbetagamma, nor is it the result of any difference in Gbetagamma binding affinity. These data suggest that myristoylation is required for specific targeting of Gpa1p to the plasma membrane, where it is needed to interact with the receptor and to regulate the release of Gbetagamma.

Base Sequence↗