title: Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.
abstract: Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Kongsoo
authors.initials: K
authors.lastname: Kang
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Michiyo
authors.initials: M
authors.lastname: Takahara
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Sakaue
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678-1297, Japan sakag@sci.u-hyogo.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D017510:Protein Folding; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology* / Q000235:genetics
chemical_list: C412459:ABCB10 protein, human; D018528:ATP-Binding Cassette Transporters; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases
keywords: endoplasmic reticulum; folding; membrane proteins; mitochondria; protein sorting signals