scholarly journals A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria

2004 ◽  
Vol 382 (1) ◽  
pp. 385-392 ◽  
Author(s):  
Abhijit MUKHOPADHYAY ◽  
Li NI ◽  
Henry WEINER
Keyword(s):  
Signal Peptide ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Major Pathway ◽  
Native Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Green Fluorescent

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDHsp–DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDHsp–DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals The N- and C-Terminal Regions of Rotavirus NSP5 Are the Critical Determinants for the Formation of Viroplasm-Like Structures Independent of NSP2

2003 ◽  
Vol 77 (22) ◽  
pp. 12184-12192 ◽  
Author(s):  
K. V. K. Mohan ◽  
J. Muller ◽  
C. D. Atreya
Keyword(s):  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Nonstructural Proteins ◽  
Binding Region ◽  
Native Form ◽  
Functional Studies ◽  
Molecular Events ◽  
C Terminus ◽  
N Terminus ◽  
Green Fluorescent

ABSTRACT Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system

2000 ◽  
Vol 113 (22) ◽  
pp. 3969-3977 ◽  
Author(s):  
A. DeRocher ◽  
C.B. Hagen ◽  
J.E. Froehlich ◽  
J.E. Feagin ◽  
M. Parsons
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Transit Peptide ◽  
Green Fluorescent ◽  
Secretory System

Apicomplexan parasites possess a plastid-like organelle called the apicoplast. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nucleus and imported post-translationally. T. gondii apicoplast proteins often have a long N-terminal extension that directs the protein to the apicoplast. It can be modeled as a bipartite targeting sequence that contains a signal sequence and a plastid transit peptide. We identified two nuclearly encoded predicted plastid proteins and made fusions with green fluorescent protein to study protein domains required for apicoplast targeting. The N-terminal 42 amino acids of the apicoplast ribosomal protein S9 directs secretion of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to direct green fluorescent protein to the apicoplast. The predicted transit peptide domain of the S9 targeting sequence directs protein to the mitochondrion in vivo. The transit peptide can also direct import of green fluorescent protein into chloroplasts in vitro. These data substantiate the model that protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.

scholarly journals Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor inSchizosaccharomyces pombe

2002 ◽  
Vol 13 (8) ◽  
pp. 2571-2584 ◽  
Author(s):  
Anjan G. Thakurta ◽  
William A. Whalen ◽  
Jin Ho Yoon ◽  
Anekella Bharathi ◽  
Libor Kozak ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Rna Recognition Motif ◽  
Synthetic Lethal ◽  
Export Activity ◽  
C Terminus ◽  
Green Fluorescent

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p inSchizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+ RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

scholarly journals In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

2008 ◽  
Vol 190 (20) ◽  
pp. 6686-6696 ◽  
Author(s):  
Yong Chang ◽  
Gary E. Wesenberg ◽  
Craig A. Bingman ◽  
Brian G. Fox
Keyword(s):  
Coenzyme A ◽  
Fluorescent Protein ◽  
Degradation Process ◽  
Side Chains ◽  
Terminal Sequence ◽  
C Terminus ◽  
A Cell ◽  
Essential Enzyme ◽  
Green Fluorescent

ABSTRACT DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Δ9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ∼10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

scholarly journals Interactions between the N- and C-termini of the mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2020 ◽  
Vol 71 (14) ◽  
pp. 4020-4032 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M Shoots ◽  
Elizabeth S Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Adaptive Responses ◽  
C Terminus ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Green Fluorescent

Abstract Although a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to green fluorescent protein (GFP) triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag, and could also be induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus could be transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.

scholarly journals PRELI (protein of relevant evolutionary and lymphoid interest) is located within an evolutionarily conserved gene cluster on chromosome 5q34-q35 and encodes a novel mitochondrial protein

2004 ◽  
Vol 378 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Sally A. STUBBS ◽  
Jimmy KYAW TUN ◽  
Jack P. LEEK ◽  
Alexander F. MARKHAM ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Subcellular Location ◽  
Protein Fusion ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Targeting Signal ◽  
Conserved Gene ◽  
Green Fluorescent

The characterization of mitochondrial proteins is important for the understanding of both normal cellular function and mitochondrial disease. In the present study we identify a novel mitochondrial protein, PRELI (protein of relevant evolutionary and lymphoid interest), that is encoded within the evolutionarily conserved MAD3/PRELI/RAB24 gene cluster located at chromosome 5q34–q35. Mouse Preli is expressed at high levels in all settings analysed; it is co-expressed with Rab24 from a strong bi-directional promoter, and is regulated independently from the S-phase-specific Mad3 gene located at its 3´ end. PRELI contains a stand-alone 170 amino acid PRELI/MSF1p´ motif at its N-terminus. This domain is found in a variety of proteins from diverse eukaryotes including yeast, Drosophila and mammals, but its function is unknown, and the subcellular location of higher eukaryotic PRELI/MSF1P´ proteins has not been determined previously. We show here that PRELI is located in the mitochondria, and by using green-fluorescent-protein fusion proteins we identify a mitochondrial targeting signal at its N-terminus.

Analysis ofChlamydomonasSF-assemblin by GFP tagging and expression of antisense constructs

2002 ◽  
Vol 115 (7) ◽  
pp. 1511-1522 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Jutta Rostmann ◽  
Andrea Grunow
Keyword(s):  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Heat Stability ◽  
Fiber Formation ◽  
Wild Type ◽  
C Terminus ◽  
Antisense Constructs ◽  
Flagellar Assembly ◽  
Green Fluorescent

Striated fiber assemblin (SF-assemblin or SFA) is the major component of the striated microtubule-associated fibers (SMAFs) in the flagellar basal apparatus of green flagellates. We generated nuclear transformants of Chlamydomonas expressing green fluorescent protein (GFP) fused to the C-terminus of SFA. SFA-GFP assembled into striated fibers that exceeded those of wild-type cells in size by several fold. At elevated temperatures(≥32°C) SFA-GFP was mostly soluble and heat shock depolymerized the SMAFs. C-terminal deletions of 18 or only six residues disturbed the ability of SFA-GFP to polymerize, indicating an important role of the C-terminal domain for fiber formation. The exchange of the penultimate Ser275 with alanine made SFA-GFP highly insoluble, causing aberrant fiber formation and conferring heat stability to the fibers. By contrast, a replacement with glutamic acid increased the solubilty of the molecule, indicating that phosphorylation on Ser275 might control solubility of SFA. In vivo observation of GFP fluorescence showed that SFA-GFP fibers were disassembled during mitosis. In cells overexpressing full-length or truncated SFA-GFP, the amount of wild-type protein was reduced. Elevated temperatures dissolved SFA-GFP fibers and induced the synthesis of SFA, suggesting that cells control both the amount of soluble and polymeric SFA. By expressing constructs consisting of cDNA and genomic DNA for parts of SFA in antiparallel configuration, the amount of SFA was severely reduced. In these strains we observed defects in flagellar assembly, indicating an important role for noncontractile striated roots in the flagella apparatus.

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