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.

Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function

1988 ◽  
Vol 8 (11) ◽  
pp. 4808-4820
Author(s):  
E M Hannig ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Normal Growth ◽  
Growth Conditions ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Noncoding Dna ◽  
Physical Description ◽  
Amino Acid Availability ◽  
Translational Activator ◽  
Starvation Conditions

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally. A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.

scholarly journals Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth conditions

2004 ◽  
Vol 3 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Christina Vessoni Penna Thereza ◽  
Ishii Marina ◽  
de Souza Luciana.Cambricoli ◽  
Cholewa Olivia
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Growth Conditions ◽  
Green Fluorescent

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 Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

2005 ◽  
Vol 187 (10) ◽  
pp. 3572-3580 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Bertolt Gust ◽  
Jolanta Zakrzewska-Czerwinska ◽  
Keith F. Chater
Keyword(s):  
Green Fluorescent Protein ◽  
Developmental Stage ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Chromosome Partitioning ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

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 Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (4) ◽  
pp. 532-538 ◽  
Author(s):  
Jacob H. Boysen ◽  
Shoba Subramanian ◽  
Aaron P. Mitchell
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Endosomal Trafficking ◽  
The Novel ◽  
Pathway Activation ◽  
Alkaline Conditions ◽  
Acidic Conditions ◽  
Green Fluorescent

ABSTRACT Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

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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