scholarly journals Peroxisomal Catalase in the Methylotrophic Yeast Candida boidinii: Transport Efficiency and Metabolic Significance

2001 ◽  
Vol 183 (21) ◽  
pp. 6372-6383 ◽  
Author(s):  
Hirofumi Horiguchi ◽  
Hiroya Yurimoto ◽  
Toh-Kheng Goh ◽  
Tomoyuki Nakagawa ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
System Analysis ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Transport Efficiency ◽  
Green Fluorescent

ABSTRACT In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Δ strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and d-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and d-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 andd-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pΔnkf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

scholarly journals Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

1990 ◽  
Vol 97 (1) ◽  
pp. 193-204 ◽  
Author(s):  
J.M. Goodman ◽  
S.B. Trapp ◽  
H. Hwang ◽  
M. Veenhuis
Keyword(s):  
Oleic Acid ◽  
Membrane Proteins ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
General Function ◽  
Amino Acid Oxidase ◽  
Peroxisomal Membrane ◽  
Methanol Metabolism

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 × 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are ‘substrate-nonspecific’. We speculate that they may relate to the structure, assembly or general function of the organelle.

Characterization and High-level Production of D-Amino Acid Oxidase in Candida boidinii

2001 ◽  
Vol 65 (3) ◽  
pp. 627-633 ◽  
Author(s):  
Hiroya YURIMOTO ◽  
Tetsuya HASEGAWA ◽  
Yasuyoshi SAKAI ◽  
Nobuo KATO
Keyword(s):  
Amino Acid ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
High Level ◽  
Level Production

scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

scholarly journals Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

2006 ◽  
Vol 5 (8) ◽  
pp. 1328-1336 ◽  
Author(s):  
Takashi Kikuma ◽  
Mamoru Ohneda ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Conidial Germination ◽  
Aerial Hyphae ◽  
Green Fluorescent ◽  
Starvation Conditions

ABSTRACT Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.

scholarly journals The C Terminus of Peripherin/rds Participates in Rod Outer Segment Targeting and Alignment of Disk Incisures

2004 ◽  
Vol 15 (4) ◽  
pp. 2027-2037 ◽  
Author(s):  
Beatrice M. Tam ◽  
Orson L. Moritz ◽  
David S. Papermaster
Keyword(s):  
Fusion Proteins ◽  
Outer Segment ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Membrane Domains ◽  
Rod Outer Segment ◽  
Retinal Degenerations ◽  
C Terminus ◽  
Green Fluorescent

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.

scholarly journals Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3)

2005 ◽  
Vol 389 (2) ◽  
pp. 355-364 ◽  
Author(s):  
Markus Napirei ◽  
Swantje Wulf ◽  
Dirk Eulitz ◽  
Hans Georg Mannherz ◽  
Thomas Kloeckl
Keyword(s):  
Fusion Proteins ◽  
Secretory Pathway ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Dnase Γ ◽  
3T3 Fibroblasts ◽  
Green Fluorescent

Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase γ, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed ‘naked’ plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.

scholarly journals A histidine-rich motif mediates mitochondrial localization of ZnT2 to modulate mitochondrial function

2011 ◽  
Vol 300 (6) ◽  
pp. C1479-C1489 ◽  
Author(s):  
Young Ah Seo ◽  
Veronica Lopez ◽  
Shannon L. Kelleher
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Mammary Glands ◽  
Mammary Cells ◽  
Mitochondrial Targeting ◽  
Mitochondrial Localization ◽  
Inner Mitochondrial Membrane ◽  
Zn Uptake ◽  
Mitochondrial Oxidation ◽  
Green Fluorescent

Female reproductive tissues such as mammary glands, ovaries, uterus, and placenta are phenotypically dynamic, requiring tight integration of bioenergetic and apoptotic mechanisms. Mitochondrial zinc (Zn) pools have emerged as a central player in regulating bioenergetics and apoptosis. Zn must first be imported into mitochondria to modulate mitochondrion-specific functions; however, mitochondrial Zn import mechanisms have not been identified. Here we documented that the Zn transporter ZnT2 is associated with the inner mitochondrial membrane and acts as an auxiliary Zn importer into mitochondria in mammary cells. We found that attenuation of ZnT2 expression significantly reduced mitochondrial Zn uptake and total mitochondrial Zn pools. Moreover, expression of a ZnT2-hemagglutinin (HA) fusion protein was localized to mitochondria and significantly increased Zn uptake and mitochondrial Zn pools, directly implicating ZnT2 in Zn import into mitochondria. Confocal microscopy of truncated and point mutants of ZnT2-green fluorescent protein (GFP) fusion proteins revealed a histidine-rich motif (51HH XH54) in the NH2 terminus that is important for mitochondrial targeting of ZnT2. More importantly, the expansion of mitochondrial Zn pools by ZnT2 overexpression significantly reduced ATP biogenesis and mitochondrial oxidation concurrent with increased apoptosis, suggesting a functional role for ZnT2-mediated Zn import into mitochondria. These results identify the first Zn transporter directly associated with mitochondria and suggest that unique secretory tissues such as the mammary gland require novel mechanisms to modulate mitochondrion-specific functions.

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