RESUMEN

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.

Asunto(s)

Proteína Coatómero/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Alelos , Membrana Celular/metabolismo , Retículo Endoplásmico/ultraestructura , Epítopos , Técnica del Anticuerpo Fluorescente Indirecta , Glutatión Transferasa/metabolismo , Aparato de Golgi/ultraestructura , Immunoblotting , Proteínas de la Membrana/metabolismo , Mutación , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fracciones Subcelulares , Temperatura , Factores de Tiempo

RESUMEN

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Asunto(s)

Proteínas de la Membrana/química , Proteínas de Transporte Vesicular , Alelos , Animales , Arginina/química , Exocitosis/fisiología , Genes Fúngicos , Glutamina/química , Sustancias Macromoleculares , Fusión de Membrana/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Microscopía Electrónica , Modelos Moleculares , Mutación , Neuronas/química , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Termodinámica

RESUMEN

Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors that influence the targeting of these proteins, alpha-factor tagged versions of Bos1p and Bet1p were employed. The alpha-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment. The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-alpha and Bet1-alpha have different cellular localization and dynamics. Bos1-alpha is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner. Bet1-alpha is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.

Asunto(s)

Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas Portadoras/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Gránulos Citoplasmáticos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de la Membrana/genética , Mutación , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/genética , Fracciones Subcelulares/química , Subtilisinas/metabolismo

RESUMEN

Ras-related, guanine nucleotide-binding proteins of the Ypt/Rab family play a key role at defined steps in vesicular transport, both in yeast and in mammalian cells. In yeast, Ypt1p has an essential function late in endoplasmic reticulum (ER) to Golgi transport, and the redundant Ypt31/Ypt32 GTPases have been proposed to act in transport through and/or from the Golgi. Here we report that mutant alleles of YPT31 and YPT32, whose gene products have a reduced affinity for GTP, are able to suppress the dominant lethal phenotype of YPT1(N121I). Co-expression of YPT1(N121I) and the suppressor YPT31(N126I) allow essentially undisturbed secretory transport in the absence of the respective wild-type GTPases. Such mutant cells massively overaccumulate 60-100 nm vesicles and are heat sensitive. It appears likely that the mutant GTPases, which are defective in nucleotide binding, compete for the binding of common interacting protein(s). These and other genetic interactions between YPT1, YPT31/32, ARF1 and SEC4 described here strongly support the view that Ypt31p and Ypt32p have a central, Golgi-associated function in anterograde or retrograde transport.

Asunto(s)

GTP Fosfohidrolasas/genética , Genes Fúngicos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab , Transporte Biológico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Genes Fúngicos/fisiología , Genes Letales , Genes Supresores , Aparato de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Mutación , Fenotipo , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Supresión Genética

RESUMEN

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

Asunto(s)

Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Membrana Celular/metabolismo , Proteína Coatómero , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microscopía Fluorescente , Proteínas Qb-SNARE , Proteínas R-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/ultraestructura

RESUMEN

Sec12p is a type II membrane glycoprotein in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae which is essential for transport vesicle budding. It is the guanine nucleotide exchange factor for the small GTP-binding protein Sar1p which is a constituent of COP II ER to Golgi vesicles. We report the sequence and localization of the human homologue to yeast Rer1p, which has recently been identified genetically as an essential component for retention of Sec12p in the ER. Reverse polymerase chain reaction was used to obtain cDNAs from HeLa cells. They code for a protein of 196 amino acids, corresponding to a molecular mass of 23 kDa. The translated sequence is 44% identical and 65% similar to yeast Rer1 protein. The four putative transmembrane domains are predicted to form a W-topology with both N- and C-terminus facing the cytosol. The functional activity of myc-tagged human Rer1 was demonstrated by the complementation of the RER1 deletion in S. cerevisiae. Mislocalization of the Sec12-reporter protein was reduced similar to the results obtained with yeast Rer1p. Human Rer1 protein was expressed in HeLa cells and the subcellular distribution analyzed by double immunofluorescence and immunoelectron microscopy of thawed cryosections. The tagged protein was localized to the Golgi apparatus and peripheral elements of the ER-Golgi interface. High overexpression leads to relocation of human Rer1 to ER-like structures together with KDEL-receptor and affects the structural organization of the Golgi apparatus. Under conditions of brefeldin A treatment, human Rer1 distributes together with recycling Golgi proteins.

Asunto(s)

Proteínas Fúngicas/genética , Aparato de Golgi/genética , Lectinas de Unión a Manosa , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Brefeldino A , Clonación Molecular , Ciclopentanos/farmacología , Prueba de Complementación Genética , Factores de Intercambio de Guanina Nucleótido , Células HeLa , Humanos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Péptidos/análisis , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Proteínas de Transporte Vesicular

RESUMEN

Saccharomyces cerevisiae MATa and MAT alpha cells secrete a-factor and alpha-factor pheromones. These peptides act on cells of the opposite mating type. They induce physiological changes which allow the formation of diploid cells. MATa strains produce an extracellular protease which cleaves, and thus inactivates the MAT alpha cell-specific alpha-factor pheromone. This pepsin-like enzyme is encoded by the BAR1(SST1) gene and is secreted into the periplasmic space of MATa cells. We found that the Bar1p protease is already active in early compartments of the secretory pathway. Our results indicate that Bar1 protease tolerates large N-terminal extensions of its substrate and does not require Golgi-specific modifications such as outer-chain glycosylation for activity.

Asunto(s)

Endopeptidasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Citosol/enzimología , Retículo Endoplásmico/enzimología , Activación Enzimática , Aparato de Golgi/enzimología , Datos de Secuencia Molecular , Proteínas R-SNARE , Receptores de Superficie Celular/metabolismo

RESUMEN

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the alpha-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.

Asunto(s)

Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteína Coatómero , Gránulos Citoplasmáticos/metabolismo , Proteínas Fúngicas/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas de la Membrana/genética , Mutación , Saccharomyces cerevisiae/ultraestructura , Proteínas de Transporte Vesicular

RESUMEN

Uso1p, a Saccharomyces cerevisiae protein required for ER to Golgi transport, is homologous to the mammalian intra-Golgi transport factor p115. We have used genetic and biochemical approaches to examine the function of Uso1p. The temperature-sensitive phenotype of the uso1-1 mutant can be suppressed by overexpression of each of the known ER to Golgi v-SNAREs (Bet1p, Bos1p, Sec22p, and Ykt6p). Overexpression of two of them, BET1p and Sec22p, can also suppress the lethality of delta uso1, indicating that the SNAREs function downstream of Uso1p. In addition, overexpression of the small GTP-binding protein Ypt1p, or of a gain if function mutant (SLY1-20) of the t-SNARE associated protein Sly1p, also confers temperature resistance. Uso1p and Ypt1p appear to function in the same process because they have a similar set of genetic interactions with the v-SNARE genes, they exhibit a synthetic lethal interaction, and they are able to suppress temperature sensitive mutants of one another when overexpressed. Uso1p acts upstream of, or in conjunction with, Ypt1p because overexpression of Ypt1p allows a delta uso1 strain to grow, whereas overexpression of Uso1p does not suppress a delta ypt1 strain. Finally, biochemical analysis indicates that Uso1p, like Ypt1p, is required for assembly of the v-SNARE/t-SNARE complex. The implications of these findings, with respect to the mechanism of vesicle docking, are discussed.

Asunto(s)

Proteínas Portadoras , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab , Secuencia de Bases , Transporte Biológico , Proteínas Fúngicas/genética , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas SNARE , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Supresión Genética

RESUMEN

Ras-related guanine nucleotide-binding proteins of the Ypt/Rab family fulfill a pivotal role in vesicular protein transport both in yeast and in mammalian cells. Proper functioning of these proteins involves their cycling between a GTP- and a GDP-bound state as well as their reversible association with specific membranes. Here we show that the yeast Ypt1 and Sec4 proteins, essential components of the vesicular transport machinery, allow unimpaired vesicular transport when permanently fixed to membranes by membrane-spanning domains replacing their two C-terminal cysteine residues. Membrane detachment of the GTPases therefore is not obligatory for transport vesicle docking to or fusion with an acceptor membrane. It was also found that the membrane anchors derived from different synaptobrevin-related proteins have targeting information and direct the chimeric GTPases to different cellular compartments, presumably from the endoplasmic reticulum via the secretory pathway.

Asunto(s)

Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab , Secuencia de Aminoácidos , Transporte Biológico , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Glicósido Hidrolasas/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Proteínas R-SNARE , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , beta-Fructofuranosidasa

RESUMEN

Mutants were isolated that are defective in the retention of a transmembrane protein in the early secretory compartments in yeast. A series of hybrid proteins was tested for their use in the selection of such mutants. Each of these hybrid proteins consisted of a type II transmembrane protein (Nin/Cout) and invertase (Suc2) as a reporter separated by a peptide linker containing a cleavage site for the Golgi protease Kex2. The integral membrane proteins which were used--Sec12p, Sec22/Sly2p or Bet1/Sly12p--are all known to be required for ER-->Golgi transport in yeast. Invertase was readily cleaved from the fusions containing Sec22/Sly2p or Bet1/Sly12p as the membrane anchoring part. In contrast, Sec12--invertase expressing transformants required mutations in either of two different genes for Kex2-dependent invertase secretion. The mutant showing the stronger retention defect (rer1) was used to clone the corresponding gene. RER1 represents the first reading frame left of the centromere of chromosome III. Cells carrying a disruption of the RER1 gene are viable and show the same mislocalizing phenotype as the original mutants. The Rer1 protein, as deduced from the nucleotide sequence, contains four transmembrane domains. It has been suggested before that Sec12p cycles between the ER and the cis-Golgi compartment. Some results obtained by using Sec12-invertase and the rer1 mutants resemble observations on the retention of Golgi-resident glycosyltransferases and viral proteins in mammalian cells. For instance, retention of Sec12-invertase is non-saturable and the membrane-spanning domain of Sec12p seems to constitute an important targeting signal.

Asunto(s)

Glicósido Hidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Orgánulos/metabolismo , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico/genética , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes Reporteros , Glicósido Hidrolasas/genética , Glicosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular , Proteínas Virales/metabolismo , beta-Fructofuranosidasa

RESUMEN

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

Asunto(s)

Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Genes Fúngicos , Genes Supresores , Aparato de Golgi/metabolismo , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab , Diploidia , Proteínas Fúngicas/aislamiento & purificación , Genotipo , Glicósido Hidrolasas/metabolismo , Haploidia , Cinética , Plásmidos , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , beta-Fructofuranosidasa

RESUMEN

In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport. By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated. Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.

Asunto(s)

Genes Fúngicos , Genes Supresores , Genes ras , Familia de Multigenes , Proteínas/genética , Receptores de Superficie Celular , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Proteínas Fúngicas/genética , Genotipo , Datos de Secuencia Molecular , Conformación Proteica , Proteínas/metabolismo , Proteínas R-SNARE , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico

RESUMEN

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C. At the restrictive temperature, ypt1ts mutants accumulated ER membranes, small vesicles, and unprocessed invertase, and they exhibited cytoskeletal defects and an enhanced 45Ca2+ uptake. Similar alterations were seen in YPT1-depleted cells. The ypt1ts mutant cells could be rescued from growth arrest by increasing extracellular Ca2+, and, even at the permissive temperature, they displayed increased trifluoperazine sensitivity.

Asunto(s)

Calcio/metabolismo , Proteínas Fúngicas/genética , Genes ras , Saccharomyces cerevisiae/genética , Proteínas ras , Alelos , Secuencia de Aminoácidos , Regulación de la Expresión Génica , Genes Fúngicos , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Supresión Genética , Temperatura

RESUMEN

Using site-directed mutagenesis, the ras-related and essential yeast YPT1 gene was changed to generate proteins with amino acid exchanges within conserved regions. Bacterially produced wild-type proteins were used for biochemical studies in vitro and were found to have properties very similar to mammalian ras proteins. Gene replacement allowed the study of physiological consequences of the mutations in yeast cells. Lys21----Met and Asn121----Ile substitutions rendered the protein incapable of binding GTP and caused lethality. Ser17----Gly and Ala65----Thr substitutions slightly changed the protein's apparent binding capacity for either GDP or GTP and altered its intrinsic GTPase activity. These mutations were without effect on cellular growth. The YPTgly17,thr65 mutant protein displayed a significantly altered relative capacity for guanine nucleotide binding but a GTPase activity comparable to the wild-type protein. In contrast to the Ala65----Thr substitution, the double mutant displayed a significantly reduced capacity for autophosphorylation and allowed cells to grow only poorly. Cellular growth was improved when this mutant protein was overproduced.

Asunto(s)

Proteínas Fúngicas/genética , Genes Fúngicos , Genes , Mutación , Saccharomyces cerevisiae/genética , Proteínas ras , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Fosforilación

RESUMEN

The 23.5 kd protein product of the ras-related YPT1 gene of S. cerevisiae was found to be essential for cell growth. The loss of YPT1 function, studied in cells with the YPT1 gene on chromosome VI regulated by the galactose-inducible GAL10 promoter, led to arrested cells that were multibudded and exhibited a complete disorganization of microtubules and an apparent loss of nuclear integrity. The YPT protein binds GTP specifically. GTP binding of the protein is essential for its intracellular function. The Asn121----IIe substitution, generated by site-directed mutagenesis, had a dominant lethal phenotype, the expression of the mutant protein led to binucleated cells and abnormal spindles. In contrast to the S. cerevisiae RAS1 and RAS2 gene products, the YPT protein seems to be involved, directly or indirectly, in microtubule organization and function.

Asunto(s)

Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas ras , Actinas/genética , Supervivencia Celular , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Genes Letales , Fenotipo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae , Transcripción Genética , Tubulina (Proteína)/genética

RESUMEN

Yeast DNA pools were prepared by ligating partial Sau3A genomic digests from strains carrying various MAL genes into the BamHI site of the yeast-Escherichia coli shuttle vector YRp7. They were used to transform recipient yeast strains that could not utilize maltose since they lacked a classical MAL gene. Transformants were obtained that could use maltose and also formed normal levels of maltase. They were unstable. They would lose the selective marker TRP1 of YRp7 alone, together with the ability to utilize maltose or only the ability to utilize maltose. The insertion of one of the plasmids was used as a hybridization probe for the others and found to share homologous sequences with all. They were then shown to contain the replication origin of the yeast 2 micron circle plasmid and additional sequences. These additional sequences were used to probe genomic digests of total yeast DNA. They hybridized at various degrees of efficiency with several bands, indicating that they were part of a family of repeated sequences. Apparently, it was the combination of the replication origin of the 2 micron circles with the additional sequences that promoted maltose utilization.

Asunto(s)

Genes Fúngicos , Maltosa/metabolismo , Saccharomyces cerevisiae/genética , Replicación del ADN , ADN de Hongos/genética , Ligamiento Genético , Plásmidos , Secuencias Repetitivas de Ácidos Nucleicos , Transformación Genética

RESUMEN

The yeast PDC1 gene coding for the fermentative enzyme pyruvate decarboxylase was isolated. This DNA sequence was used to identify the corresponding messenger RNA by hybridization. It could be shown that the synthesis of pyruvate decarboxylase is efficiently regulated by variations in the amount of PDC1 mRNA. Very low levels of PDC1 mRNA were found in cells growing in a medium containing ethanol. Glucose addition to these cells leads to a rapid accumulation of PDC1 mRNA. The PDC1 mRNA levels found in different mutants and in cells growing in media containing carbon sources other than glucose or ethanol suggest that the amount of PDC1 mRNA in yeast cells is affected by a number of different factors.

Asunto(s)

Carboxiliasas/biosíntesis , Piruvato Descarboxilasa/biosíntesis , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Genes , Genes Fúngicos , Piruvato Descarboxilasa/genética , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética

RESUMEN

Six different pyruvate decarboxylase mutants of Saccharomyces cerevisiae were isolated. They belong to two unlinked complementation groups. Evidence is presented that one group is affected in a structural gene. The fact that five of the six mutants had residual pyruvate decarboxylase activity provided the opportunity for an intensive physiological characterization. It was shown that the loss of enzyme activity in vitro is reflected in a lower fermentation rate, an increased pyruvate secretion, and slower growth on a 2% glucose medium. The different effects of antimycin A on leaky mutants grown on ethanol versus the same mutants grown on glucose support the view that glucose induces some of the glycolytic enzymes, especially pyruvate decarboxylase.

Asunto(s)

Carboxiliasas/genética , Genes , Glucólisis , Piruvato Descarboxilasa/genética , Saccharomyces cerevisiae/enzimología , Antimicina A/farmacología , Prueba de Complementación Genética , Glucosa/metabolismo , Mutación , Piruvato Descarboxilasa/metabolismo , Piruvatos/metabolismo , Ácido Pirúvico , Saccharomyces cerevisiae/genética