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1.
Traffic ; 15(12): 1344-65, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25243755

ABSTRACT

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.


Subject(s)
Adenosine Triphosphatases/metabolism , Cation Transport Proteins/metabolism , Copper/metabolism , Endosomes/metabolism , Hepatocytes/metabolism , Animals , Cell Line, Tumor , Copper-Transporting ATPases , Hepatocytes/drug effects , Macrolides/pharmacology , Protein Transport , Rats , trans-Golgi Network/metabolism
2.
Mol Biol Cell ; 14(7): 2689-705, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12857857

ABSTRACT

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.


Subject(s)
Cholesterol/metabolism , Endosomes/metabolism , Glycosphingolipids/metabolism , Hepatocytes/metabolism , Animals , Biological Transport/physiology , Cell Membrane/metabolism , Cell Membrane/physiology , Cell Polarity/physiology , Cells, Cultured , Cholesterol/physiology , Cloning, Molecular , Detergents/pharmacology , Endocytosis/physiology , Endosomes/physiology , Glycosphingolipids/physiology , Golgi Apparatus/metabolism , Golgi Apparatus/physiology , Hepatocytes/physiology , Membrane Microdomains/metabolism , Membrane Microdomains/physiology , Rats , Solubility/drug effects , trans-Golgi Network/metabolism , trans-Golgi Network/physiology
3.
Mol Biol Cell ; 13(10): 3400-15, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12388745

ABSTRACT

Membrane trafficking is central to establishing and maintaining epithelial cell polarity. One open question is to what extent the mechanisms regulating membrane trafficking are conserved between nonpolarized and polarized cells. To answer this question, we examined the dynamics of domain-specific plasma membrane (PM) proteins in three classes of hepatic cells: polarized and differentiated WIF-B cells, nonpolarized and differentiated Fao cells, and nonpolarized and nondifferentiated Clone 9 cells. In nonpolarized cells, mature apical proteins were uniformly distributed in the PM. Surprisingly, they were also in an intracellular compartment. Double labeling revealed that the compartment contained only apical proteins. By monitoring the dynamics of antibody-labeled molecules in nonpolarized cells, we further found that apical proteins rapidly recycled between the compartment and PM. In contrast, the apical PM residents in polarized cells showed neither internalization nor return to the basolateral PM from which they had originally come. Cytochalasin D treatment of these polarized cells revealed that the retention mechanisms are actin dependent. We conclude from these data that both polarized and nonpolarized cells selectively sort apical proteins from the PM and transport them to specific, but different cellular locations. We propose that the intracellular recycling compartment in nonpolarized cells is an intermediate in apical surface formation.


Subject(s)
Cell Membrane/metabolism , Cell Polarity , Hepatocytes/metabolism , Membrane Proteins/metabolism , Protein Transport/physiology , Actins/metabolism , Animals , Biomarkers , Cell Compartmentation , Cell Line , Hepatocytes/cytology , Models, Biological , Protein Structure, Tertiary , Rats
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