RESUMEN
Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs-the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p-interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.
Asunto(s)
Endorribonucleasas , Proteínas Asociadas a Microtúbulos , Mitosis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sumoilación , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.
Asunto(s)
Áfidos/metabolismo , Proteínas Bacterianas/metabolismo , Buchnera/metabolismo , Proteínas de Insectos/metabolismo , Proteoma/metabolismo , Algoritmos , Aminoácidos/metabolismo , Animales , Áfidos/enzimología , Áfidos/microbiología , Buchnera/enzimología , Fraccionamiento Celular , Chaperonina 60/metabolismo , Análisis por Conglomerados , Gluconeogénesis , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas , Pisum sativum , Purinas/metabolismo , Análisis de Secuencia de Proteína , Simbiosis , Espectrometría de Masas en TándemRESUMEN
Aß(1-40) coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aß (a hallmark of Alzheimer's disease). We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aß aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.