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Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway.

ABSTRACT: To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrP(c)) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrP(c) internalization via the constitutive endocytic pathway in the absence of Cu(2+) and the stimulated pathway in the presence of Cu(2+) were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrP(c) either in the presence or absence of Cu(2+), in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrP(c) was inhibited by the raft-disrupting drugs filipin and nystatin, and by the dominant-negative dynamin-1 mutant dynamin-1 K44A, both in the presence and absence of Cu(2+). The internalized PrP(c) was found to colocalize with cargo that traffic in the Arf6 pathway and in large vacuoles in cells expressing the Arf6 dominant-active mutant. These results show that PrP(c) is internalized in a clathrin-independent pathway that is associated with Arf6.

SUBMITTER: Kang YS

PROVIDER: S-EPMC2798124 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway.

Kang Young-Shin YS Zhao Xiaohong X Lovaas Jenna J Eisenberg Evan E Greene Lois E LE

Journal of cell science 20091020 Pt 22

To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrP(c)) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrP(c) internalization via the constitutive endocytic pathway in the absence of Cu(2+) and the stimulated pathway in the presence of Cu(2+) were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrP(c) either in the presence or ab ...[more]

PMID: 19843586

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Project description:BackgroundTriacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.ResultsAlthough LRP in these cells is fully active as established by substantial alpha2macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K+, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.ConclusionThese data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

Dataset Information

Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway.

Publications

Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway.

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