Visualization of Mitochondrial Protein Import in Cultured Mammalian Cells with Green Fluorescent Protein and Effects of Overexpression of the Human Import Receptor Tom20

Abstract We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

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Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

Journal of Cell Science ◽

10.1242/jcs.112.16.2705 ◽

1999 ◽

Vol 112 (16) ◽

pp. 2705-2714

Author(s):

E.M. Burns ◽

L. Christopoulou ◽

P. Corish ◽

C. Tyler-Smith

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Quantitative Measurement ◽

Fluorescent Protein ◽

Cultured Cells ◽

Chromosome Loss ◽

Facs Analysis ◽

Fluorescent Cells ◽

Green Fluorescent ◽

Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

Immunology Letters ◽

10.1016/j.imlet.2017.12.005 ◽

2018 ◽

Vol 194 ◽

pp. 29-39 ◽

Cited By ~ 10

Author(s):

Fatemeh Motevalli ◽

Azam Bolhassani ◽

Shilan Hesami ◽

Sepideh Shahbazi

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Green Fluorescent

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Mitochondrial assembly: protein import

Proceedings of The Nutrition Society ◽

10.1079/pns2004342 ◽

2004 ◽

Vol 63 (2) ◽

pp. 293-300 ◽

Cited By ~ 26

Author(s):

David A. Hood ◽

Anna-Maria Joseph

Keyword(s):

Molecular Chaperones ◽

Mammalian Cells ◽

Protein Import ◽

Contractile Activity ◽

Mitochondrial Protein ◽

Energy Status ◽

Mitochondrial Protein Import ◽

Physiological Importance ◽

The Matrix ◽

Import Receptors

The protein import process of mitochondria is vital for the assembly of the hundreds of nuclear-derived proteins into an expanding organelle reticulum. Most of our knowledge of this complex multisubunit network comes from studies of yeast and fungal systems, with little information known about the protein import process in mammalian cells, particularly skeletal muscle. However, growing evidence indicates that the protein import machinery can respond to changes in the energy status of the cell. In particular, contractile activity, a powerful inducer of mitochondrial biogenesis, has been shown to alter the stoichiometry of the protein import apparatus via changes in several protein import machinery components. These adaptations include the induction of cytosolic molecular chaperones that transport precursors to the matrix, the up-regulation of outer membrane import receptors, and the increase in matrix chaperonins that facilitate the import and proper folding of the protein for subsequent compartmentation in the matrix or inner membrane. The physiological importance of these changes is an increased capacity for import into the organelle at any given precursor concentration. Defects in the protein import machinery components have been associated with mitochondrial disorders. Thus, contractile activity may serve as a possible mechanism for up-regulation of mitochondrial protein import and compensation for mitochondrial phenotype alterations observed in diseased muscle.

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A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants

Biochemical Journal ◽

10.1042/bj20021966 ◽

2003 ◽

Vol 373 (2) ◽

pp. 403-408 ◽

Cited By ~ 67

Author(s):

Nadya G. GURSKAYA ◽

Arkady F. FRADKOV ◽

Natalia I. POUNKOVA ◽

Dmitry B. STAROVEROV ◽

Maria E. BULINA ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Random Mutagenesis ◽

Wild Type ◽

Green Luminescence ◽

Mammalian Cell Lines ◽

Biological Tool ◽

Fluorescent Pattern ◽

Green Fluorescent

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

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Interdependence of the Peroxisome-targeting Receptors in Arabidopsis thaliana: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes

Molecular Biology of the Cell ◽

10.1091/mbc.e09-08-0672 ◽

2010 ◽

Vol 21 (7) ◽

pp. 1263-1271 ◽

Cited By ~ 31

Author(s):

Naxhiely Martínez Ramón ◽

Bonnie Bartel

Keyword(s):

Arabidopsis Thaliana ◽

Green Fluorescent Protein ◽

Protein Import ◽

Fluorescent Protein ◽

Protein Accumulation ◽

Animal Development ◽

Metabolic Reactions ◽

Targeting Signals ◽

Green Fluorescent ◽

Peroxisome Targeting Signals

Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.

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High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells

Blood ◽

10.1182/blood.v90.9.3316 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3316-3321 ◽

Cited By ~ 53

Author(s):

Ana Limón ◽

Javier Briones ◽

Teresa Puig ◽

Mercé Carmona ◽

Oscar Fornas ◽

...

Keyword(s):

Flow Cytometry ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Hematopoietic Cells ◽

Retroviral Vectors ◽

Green Fluorescent Protein Gene ◽

Efficient System ◽

Green Fluorescent

Abstract Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34+ cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

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