Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells.
Bottom Line: Apolipoprotein A-I (ApoA-I) is an extracellular lipid acceptor, whose role in cholesterol efflux and high-density lipoprotein formation is mediated by ATP-binding cassette transporter A1 (ABCA1).Heart amyloid fibrils were found to be mainly constituted by the 93-residue N-terminal fragment of ApoA-I, named [1-93]ApoA-I.We provide evidence that the polypeptide: (i) binds to specific sites on cell membrane (K(d) = 5.90 ± 0.70 × 10(-7) M), where it partially co-localizes with ABCA1, as also described for ApoA-I; (ii) is internalized mostly by chlatrin-mediated endocytosis and lipid rafts, whereas ApoA-I is internalized preferentially by chlatrin-coated pits and macropinocytosis and (iii) is rapidly degraded by proteasome and lysosomes, whereas ApoA-I partially co-localizes with recycling endosomes.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.Show MeSH
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Mentions: According to recent reports, ApoA-I, once internalized, is recycled back to the cell surface [3–5]. To investigate the retroendocytosis pathway, Rab4 was used as a marker, as it is located in vesicles directing protein recycling from early endosomes to the plasma membrane. We transiently transfected H9c2 cardiomyoblasts with a vector encoding Rab4 fused to the GFP. Twenty-four hours after transfection, cells were incubated with rhodamine-[1–93]ApoA-I, or ApoA-I, for 6 hrs at 37°C. As shown in Fig. 5A, [1–93]ApoA-I (red) does not co-localize with Rab4+ endosomal compartments (green), whereas significant signals of co-localization were observed for ApoA-I (Fig. 5B). Taken together, these results clearly indicate that the fibrillogenic polypeptide, once internalized in cardiomyoblasts, is not recycled to the cell membrane, whereas ApoA-I is shuttled back to the plasma membrane to be resecreted, as described for other cell lines [3–5].
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.