Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane

2010 ◽  
Vol 37 (8) ◽  
pp. 785 ◽  
Author(s):  
Mark A. Jackson ◽  
Kerry A. Nutt ◽  
Rachael Hassall ◽  
Anne L. Rae
Keyword(s):  
Endoplasmic Reticulum ◽  
Limited Proteolysis ◽  
Subcellular Location ◽  
Pathogen Resistance ◽  
Careful Consideration ◽  
In Planta ◽  
Subcellular Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Lytic Vacuole

Transgenic sugarcane plants (Saccharum hybrid) have been proposed as a production platform for recombinant proteins, including those providing pathogen resistance as well as high value therapeutic proteins. For the in planta production of proteins that are potentially toxic, a careful consideration of subcellular location is required in order to optimise yield and to avoid detrimental interaction with plant cellular processes. In this study, avidin, a glycoprotein that is potentially toxic to cells because of its high affinity to the co-vitamin biotin, was used to test the effectiveness of a range of targeting signals. Accumulation of avidin was directed to the apoplast, endoplasmic reticulum and to the lytic and delta type vacuoles. Although targeting to the delta vacuole resulted in the highest yields of avidin, these plants developed a biotin deficient phenotype, indicating that this targeting was not fully effective in protecting cellular biotin pools. Similar problems were also observed when avidin was retained in the endoplasmic reticulum. When avidin was targeted to the lytic vacuole using the targeting signal from the sugarcane legumain, plants remained phenotypically normal; however, avidin was predominantly detected as a degraded product due to site-specific limited proteolysis in the vacuole. For avidin and other potentially toxic products, this lytic vacuole targeting signal may be useful if stability within this proteolytic environment can be improved.

scholarly journals Protein Import into Hydrogenosomes of Trichomonas vaginalis Involves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases

2008 ◽  
Vol 7 (10) ◽  
pp. 1750-1757 ◽  
Author(s):  
Marek Mentel ◽  
Verena Zimorski ◽  
Patrick Haferkamp ◽  
William Martin ◽  
Katrin Henze
Keyword(s):  
Trichomonas Vaginalis ◽  
Protein Import ◽  
Atp Synthesis ◽  
Antioxidant Systems ◽  
Low Oxygen ◽  
Oxygen Sensitive ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Thioredoxin Reductases

ABSTRACT The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H2-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.

scholarly journals Identification of Novel Plant Peroxisomal Targeting Signals by a Combination of Machine Learning Methods and in Vivo Subcellular Targeting Analyses

2011 ◽  
Vol 23 (4) ◽  
pp. 1556-1572 ◽  
Author(s):  
Thomas Lingner ◽  
Amr R. Kataya ◽  
Gerardo E. Antonicelli ◽  
Aline Benichou ◽  
Kjersti Nilssen ◽  
...  
Keyword(s):  
Machine Learning ◽  
Subcellular Targeting ◽  
Learning Methods ◽  
Machine Learning Methods ◽  
Peroxisomal Targeting ◽  
Targeting Signals

Dual targeting ability of targeting signals is dependent on the nature of the mature protein

2003 ◽  
Vol 30 (7) ◽  
pp. 805 ◽  
Author(s):  
Orinda Chew ◽  
James Whelan
Keyword(s):  
Alternative Oxidase ◽  
Small Subunit ◽  
Trna Synthetase ◽  
Mature Protein ◽  
Bisphosphate Carboxylase ◽  
Dual Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Targeting Ability ◽  
Passenger Protein

The targeting ability of three signals previously shown to support the import of passenger proteins into both mitochondria and chloroplasts was investigated with authentic mitochondrial or chloroplastic proteins. An in vitro dual import assay that maintained import specificity showed that the ability of dual signals to support mitochondrial and chloroplastic import depended on the nature of the passenger protein. All dual targeting signals supported import of their native mature protein as a passenger into both mitochondria and chloroplasts. However the glutathione reductase targeting signal only supported mitochondrial import with the mitochondrial protein alternative oxidase, and chloroplast import with the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase. The Arabidopsis histidyl-tRNA synthetase targeting signal only supported mitochondrial import with the alternative oxidase as a passenger, but the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase was imported into both mitochondria and chloroplasts. The Arabidopsis asparaginyl-tRNA synthetase supported import of alternative oxidase and the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase into both mitochondria and chloroplasts. Analysis of the targeting signals of all known dual targeted proteins using targeting predictions indicates that most of them are more strongly predicted to be chloroplast-targeted. Secondary structure predictions indicate the ability of most dual targeted signals to form both α-helical and β-sheet-type structures, a feature of mitochondrial and plastid targeting signals, respectively. Thus, it appears that a major determinant of dual targeting ability is the nature of the mature or passenger protein.

scholarly journals An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin domain and the human cytokine interleukin 6 is efficiently cleaved in planta

2013 ◽  
Vol 12 (3) ◽  
pp. 311-319
Author(s):  
Nausch Henrik ◽  
Mikschofsky Heike ◽  
Koslowski Roswitha ◽  
Steinmann Alain ◽  
Meyer Udo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Interleukin 6 ◽  
In Planta ◽  
Human Cytokine

scholarly journals Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity

1983 ◽  
Vol 213 (1) ◽  
pp. 99-105 ◽  
Author(s):  
S R Wilson ◽  
M D Houslay
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteinase Inhibitors ◽  
Smooth Endoplasmic Reticulum ◽  
Limited Proteolysis ◽  
Proteolytic Activation ◽  
Freeze Thaw ◽  
Membrane Bound ◽  
Time Courses ◽  
Triton X ◽  
Thiol Proteinase

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.

scholarly journals Expression of a Gene for Cyclophilin Which Contains an Amino-Terminal Endoplasmic Reticulum-Targeting Signal

1999 ◽  
Vol 40 (1) ◽  
pp. 77-87 ◽  
Author(s):  
T. Saito ◽  
Y. Niwa ◽  
H. Ashida ◽  
K. Tanaka ◽  
M. Kawamukai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Terminal ◽  
Targeting Signal ◽  
Endoplasmic Reticulum Targeting

scholarly journals The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors

2008 ◽  
Vol 183 (3) ◽  
pp. 485-498 ◽  
Author(s):  
Sheila A. Baker ◽  
Mohammad Haeri ◽  
Peter Yoo ◽  
Sidney M. Gospe ◽  
Nikolai P. Skiba ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Outer Segment ◽  
The Other ◽  
Visual Signals ◽  
Its Sequence ◽  
Specific Targeting ◽  
New Material ◽  
Targeting Signals ◽  
Syntaxin 3

Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals are confined to the outer segment. Yet, the mechanisms responsible for establishing and maintaining photoreceptor compartmentalization are poorly understood. Here we investigated the targeting of two related membrane proteins, R9AP and syntaxin 3, one residing within and the other excluded from the outer segment. Surprisingly, we have found that only syntaxin 3 has targeting information encoded in its sequence and its removal redirects this protein to the outer segment. Furthermore, proteins residing in the endoplasmic reticulum and mitochondria were similarly redirected to the outer segment after removing their targeting signals. This reveals a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. This also implies that every protein residing outside the outer segment must have a means to avoid this “default” trafficking flow.

scholarly journals Control of protein trafficking by reversible masking of transport signals

2016 ◽  
Vol 27 (8) ◽  
pp. 1310-1319 ◽  
Author(s):  
Omer Abraham ◽  
Karnit Gotliv ◽  
Anna Parnis ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Protein Import ◽  
Binding Peptide ◽  
Protein Traffic ◽  
Alternative Approach ◽  
Targeting Signal ◽  
Regulated Trafficking ◽  
Streptavidin Binding

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

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