NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells.
Cu is an essential cofactor of cellular proteins but is toxic in its free state. The hepatic Cu-ATPase ATP7B has two functions in Cu homeostasis: it loads Cu+ onto newly synthesized apoceruloplasmin in the secretory pathway, thereby activating the plasma protein; and it participates in the excretion of excess Cu+ into the bile. To carry out these two functions, the membrane protein responds to changes in intracellular Cu levels by cycling between the Golgi and apical region. We used polarized hepatic WIF-B cells and high-resolution confocal microscopy to map the itinerary of endogenous and exogenous ATP7B under different Cu conditions. In Cu-depleted cells, ATP7B resided in a post-trans-Golgi network compartment that also contained syntaxin 6, whereas in Cu-loaded cells, the protein relocated to unique vesicles very near to the apical plasma membrane as well as the membrane itself. To determine the role of ATP7B's cytoplasmic NH2 terminus in regulating its intracellular movements, we generated seven mutations/deletions in this large [approximately 650 amino acid (AA)] domain and analyzed the Cu-dependent behavior of the mutant ATP7B proteins in WIF-B cells. Truncation of the ATP7B NH2 terminus up to the fifth copper-binding domain (CBD5) yielded an active ATPase that was insensitive to cellular Cu levels and constitutively trafficked to the opposite (basolateral) plasma membrane domain. Fusion of the NH2-terminal 63 AA of ATP7B to the truncated protein restored both its Cu responsiveness and correct intracellular targeting. These results indicate that important targeting information is contained in this relatively short sequence, which is absent from the related CuATPase, ATP7A.
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Y
Y
Guo
L
L
Nyasae
L T
LT
Braiterman
A L
AL
Hubbard
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D027682:Cation Transport Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases