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1.
J Biol Chem ; 276(39): 36501-7, 2001 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-11443138

RESUMEN

Most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the membrane. We have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the consensus sequence, EXXYXQ downward arrowS (tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are selectively active on soluble cytosolic and peroxisomal tev-containing proteins in vivo, respectively, without affecting the viability of the yeast cells. The tev sequence was introduced in between the primary sequence of the peroxisomal membrane proteins Pex3p or Pex10p and the reporter protein enhanced green fluorescent protein (eGFP). Co-synthesis of these functional tev-GFP tagged proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed that the C termini of Pex3p and Pex10p are exposed to the cytosol. Additional applications of the TEV protease to study peroxisome biogenesis are discussed.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Bioquímica , Endopeptidasas/química , Membranas Intracelulares/química , Peroxisomas/química , Proteínas de Saccharomyces cerevisiae , Fenómenos Bioquímicos , Western Blotting , Citosol/enzimología , Proteínas Fúngicas/química , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/química , Peroxinas , Peroxisomas/enzimología , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/metabolismo
2.
FEMS Yeast Res ; 1(1): 23-31, 2001 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12702460

RESUMEN

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.


Asunto(s)
Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Glucosa/metabolismo , Nitrógeno/metabolismo , Peroxisomas/metabolismo , Pichia/metabolismo , Autofagia , Proteínas Fúngicas/genética , Metanol/metabolismo , Microscopía Electrónica , Pichia/genética , Pichia/crecimiento & desarrollo
3.
J Biol Chem ; 275(14): 9986-95, 2000 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-10744674

RESUMEN

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg(11), Lys(13), Lys(14), or Lys(15) with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Consenso , Secuencia Conservada , Cartilla de ADN , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes , Membranas Intracelulares/ultraestructura , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Peroxinas , Peroxisomas/ultraestructura , Pichia/genética , Pichia/crecimiento & desarrollo , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido
4.
Biochem J ; 341 ( Pt 3): 777-84, 1999 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-10417344

RESUMEN

Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.


Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Citosol/enzimología , Mitocondrias/enzimología , Secuencia de Aminoácidos , Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/genética , Citosol/ultraestructura , Endocitosis , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , Proteínas Luminiscentes/genética , Microscopía Electrónica , Mitocondrias/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
5.
FEBS Lett ; 411(1): 133-9, 1997 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-9247158

RESUMEN

We have studied the effect of brefeldin A (BFA), a fungal toxin that interferes with coated vesicle formation, on the biogenesis of peroxisomes in the yeast Hansenula polymorpha. Addition of BFA (20 microg/ml) to cultures of H. polymorpha partially inhibited the development of peroxisomes and resulted in the reversible accumulation of newly synthesized peroxisomal membrane and matrix proteins at the endoplasmic reticulum. In contrast, BFA did not interfere with the selective degradation of peroxisomes. Taken together, our data suggest that the ER plays a crucial role in peroxisome biogenesis in H. polymorpha, possibly in the biosynthesis of the peroxisomal membrane.


Asunto(s)
Ciclopentanos/farmacología , Microcuerpos/efectos de los fármacos , Pichia/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte Biológico , Brefeldino A , Retículo Endoplásmico/metabolismo , Glucosa/farmacología , Microcuerpos/metabolismo , Pichia/crecimiento & desarrollo , Pichia/metabolismo
6.
Plant Physiol ; 101(1): 237-243, 1993 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12231679

RESUMEN

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.

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