Methotrexate does not block import of a DHFR fusion protein into chloroplasts

America, Twan; Hageman, Johan; Keegstra, Kenneth; Weisbeek, Peter

doi:10.1007/978-94-009-0383-8_37

Cited by 17 publications

(27 citation statements)

References 7 publications

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“…Indeed, translocation of proteins into the ER and mitochondria is based upon their unfolded conformation [1, 2]. To elucidate whether the nonclassical export of FGF1 requires unfolding of FGF1 and S100A13, we applied the method of DHFR tagging of the released proteins [45]. FGF1 and S100A13, a member of both the FGF1 and IL1α release complexes, were C-terminally tagged with DHFR (Figure 1A), which is an enzyme locked in folded conformation by its specific inhibitor AP [45].…”

Section: Resultsmentioning

confidence: 99%

“…To elucidate whether the nonclassical export of FGF1 requires unfolding of FGF1 and S100A13, we applied the method of DHFR tagging of the released proteins [45]. FGF1 and S100A13, a member of both the FGF1 and IL1α release complexes, were C-terminally tagged with DHFR (Figure 1A), which is an enzyme locked in folded conformation by its specific inhibitor AP [45]. FGF1:DHFR and 6Myc:S100A13:DHFR chimeras were transfected to NIH 3T3 cells and their expression was verified by using, respectively anti-FGF1, and anti-Myc antibodies (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Studies based on the production of dihydrofolate reductase (DHFR)-containing chimeras are conducted to clarify the role of unfolding in protein translocation across the biological membranes [45]. This method is based on the use of a specific DHFR inhibitor aminopterin (AP) that stabilizes the tertiary structure of the enzyme or its chimeras, and prevents their transport across biological membranes if this transport requires protein unfolding [45].…”

Section: Introductionmentioning

confidence: 99%

“…This method is based on the use of a specific DHFR inhibitor aminopterin (AP) that stabilizes the tertiary structure of the enzyme or its chimeras, and prevents their transport across biological membranes if this transport requires protein unfolding [45]. Using this method, Nickel et al demonstrated that FGF2 does not require total unfolding for its unconventional export [7].…”

Section: Introductionmentioning

confidence: 99%

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Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Graziani

Doyle

Sterling

et al. 2009

Biochemical and Biophysical Research Communications

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Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Graziani

Doyle

Sterling

et al. 2009

Biochemical and Biophysical Research Communications

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“…These proteins were competent for import probably due to unfoldases activity presented in the envelope (Guera et al 1993). The import machinery of chloroplast envelope can unfold even tightly folded protein domains imported into chloroplast (America et al 1994, Endo et al 1994, Walker et al 1996. The study with mitochondria shows that the import of DHFR (dihydrofolate reductase) domain attached to the C-terminus of the mitochondrial precursor protein is blocked by the presence of methotrexat (Eilers and Schatz 1986).…”

Section: Transport Of Protein Across the Chloroplast Envelopementioning

confidence: 99%

Targeting of proteins into and within the chloroplast

et al. 1998

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Functional Analysis of Semi-conserved Transit Peptide Motifs and Mechanistic Implications in Precursor Targeting and Recognition

et al. 2016

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Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide (TP). Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer (TOC) and inner envelope (TOC). The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semi-conserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit (SStp) and ferredoxin (Fdtp). Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations (∼85) in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.

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Methotrexate does not block import of a DHFR fusion protein into chloroplasts

Cited by 17 publications

References 7 publications

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Targeting of proteins into and within the chloroplast

Functional Analysis of Semi-conserved Transit Peptide Motifs and Mechanistic Implications in Precursor Targeting and Recognition

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