Size limitation in translocation of fibroblast growth factor 1 fusion proteins across the endosomal membrane

Malgorzata Zakrzewska, Yan Zhen, Antoni G Wiedlocha, Sjur Olsnes, Jorgen Wesche

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

After binding to its receptor on the surface of mammalian cells and subsequent endocytosis, FGF1 is translocated across the membrane into the cytosol. The growth factor is then further transported into the nucleus. In order to characterize more closely the translocation mechanism utilized by FGF1, we introduced additional amino acids into FGF1 to test the size dependence of the translocated substrate. We constructed mutants containing an increasing number of copies of the myc tag (1-13 copies) in a surface loop of the FGF1 molecule. All of the constructs bound to specific FGF receptors and to heparin and were taken up by endocytosis. However, only FGF1 mutants harboring up to three myc tags (53 amino acids) were translocated while mutants with five myc tags (77 amino acids) or more were not translocated through the membrane. We further showed that insertion of other, unrelated polypeptides into FGF1, i.e., 3 x FLAG tag (22 amino acids) and streptavidin binding peptide (50 amino acids), was also translocated. Larger insertions into FGF1, like the CBP-SBP tag (82 amino acids) or ricin A-chain (272 amino acids), resulted in fusion proteins that failed to translocate. The presented data imply that it is possible to employ FGF1 to import various polypeptides into the cytosol and nucleus of cells. Furthermore, the strict size dependence of FGF1 fusion proteins in membrane translocation argues against simple leakage of FGF1 from ruptured endosomal membranes but rather points to a specific translocation apparatus involving a proteinaceous pore.

Original languageEnglish (US)
Pages (from-to)7209-7218
Number of pages10
JournalBiochemistry
Volume48
Issue number30
DOIs
StatePublished - Aug 4 2009

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Fibroblast Growth Factor 1
Fusion reactions
Membranes
Amino Acids
Proteins
Endocytosis
Cytosol
Membrane Fusion Proteins
Ricin
Fibroblast Growth Factor Receptors
Peptides
Cell Nucleus
Heparin
Intercellular Signaling Peptides and Proteins
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Size limitation in translocation of fibroblast growth factor 1 fusion proteins across the endosomal membrane. / Zakrzewska, Malgorzata; Zhen, Yan; Wiedlocha, Antoni G; Olsnes, Sjur; Wesche, Jorgen.

In: Biochemistry, Vol. 48, No. 30, 04.08.2009, p. 7209-7218.

Research output: Contribution to journalArticle

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