Visualization of Mitochondrial Protein Import in Cultured Mammalian Cells with Green Fluorescent Protein and Effects of Overexpression of the Human Import Receptor Tom20

Abstract

The presequence of the ornithine transcarbamylase precursor (pOTC) was fused to green fluorescent protein (GFP), yielding pOTC-GFP and pOTCN-GFP containing the presequence plus 4 and 58 residues of mature ornithine transcarbamylase, respectively. When GFP cDNA was transfected into COS-7 cells, the cytosol and nucleus were fluorescent. On the other hand, pOTC-GFP cDNA gave strong fluorescence of a unique mitochondrial pattern. After fractionation of cells expressing pOTC-GFP with digitonin, fluorescence was recovered mostly in the particulate fraction. Immunoblot analysis showed that processed GFP was present in the particulate fraction, whereas pOTC-GFP was recovered in both the soluble and particulate fractions. pOTC-GFP and pOTCN-GFP synthesized in vitro were imported efficiently into the isolated mitochondria. Single and triple amino acid mutations in the presequence resulted in impaired mitochondrial import and in a loss of mitochondrial fluorescence. Perinuclear aggregation of fluorescent mitochondria was observed when the human mitochondrial import receptor Tom20 (hTom20) was coexpressed with pOTC-GFP. Overexpression of hTom20 (not ⌬hTom20, which lacks the anchor sequence) resulted in stimulated mitochondrial import of pOTC-GFP in COS-7 cells. When pOTC-GFP cDNA was microinjected into nuclei of human fibroblast cells, mitochondrial fluorescence was detected as early as 2-3 h after injection. These results show that GFP fusion protein can be used to visualize mitochondrial structures and to monitor mitochondrial protein import in a single cell in real time.

Targeting Proteins to the Lumen of Endoplasmic Reticulum Using N-terminal Domains of 11β-Hydroxysteroid Dehydrogenase and the 50-kDa Esterase

“…Expression of native GFP showed green fluorescent signals in both the cytoplasm and nucleus (Fig. 2B) consistent with the previous studies (28). To explore whether the N-terminal domain of E3 also imparts GFP a membrane localization, GFP constructs hum and rab E3N-GFP (Constructs c and e, respectively) were generated and expressed in COS-7 cells.…”

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

“…The PCR fragment was digested with XhoI, and cloned into the same site of pCAGGS yielding pCAGGS-ChMTX. Construction of pCAGGS-hTom20 (26), pCAGGS-pOTC (22), pCAGGS-pOTC-GFP (22) and pCAGGS-hAII (27) were described previously.…”

“…Proteins were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Antisera against human OTC (20), gelly fish GFP (22), human arginase II (29), hTom20 (22), human Tom22 (30), Tom40, and human metaxin were used as primary antibodies. Immunodetection was performed by the use of the chemiluminescence kit (ECL kit, Amersham).…”

Human DnaJ Homologs dj2 and dj3, and bag-1 Are Positive Cochaperones of hsc70

“…We then asked whether dj2 and dj3 are also required for other precursor proteins where the requirement for hsc70 varies by 0 -60% for import. When import of human pOTC, pre-aspartate aminotransferase, pre-serine:pyruvate aminotransferase, and an artificial fusion precursor protein (27) in which the presequence of human pOTC was fused to green fluorescence protein (pOTC-GFP) was tested, dependence on hsc70 roughly paralleled that on dj2-dj3 among the precursor proteins (data not shown).…”