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J. Biol. Chem., Vol. 275, Issue 21, 15917-15925, May 26, 2000
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From the Department of Physiology, Tufts University School of
Medicine, Boston, Massachusetts 02111
Newly synthesized canalicular ectoenzymes and a
cell adhesion molecule (cCAM105) have been shown to traffic from the
Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all
canalicular proteins are targeted via this indirect route in
hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [35S]methionine metabolic
labeling followed by preparation of highly purified Golgi membranes and
canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles
and subsequent immunoprecipitation. In particular, we compared membrane
targeting of newly synthesized canalicular ABC
(ATP-binding cassette) transporters
MDR1, MDR2, and SPGP (sister of
P-glycoprotein) with that of
cCAM105. Significant differences were observed in metabolic pulse-chase
labeling experiments with regard to membrane targeting of these apical
proteins. After a chase time of 15 min, cCAM105 appeared exclusively in
SMVs, peaked at 1 h, and progressively declined thereafter. In
CMVs, cCAM105 was first detected after 1 h and subsequently
increased for 3 h. This findings confirm the transcytotic
targeting of cCAM105 reported in earlier studies. In contrast, at no
time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In
CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after
2 h of labeling. In Golgi membranes, each of the ABC transporters
peaked at 30 min and was virtually absent thereafter. These data
suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in
an intracellular pool en route from the Golgi to the apical plasma
membrane. This study provides biochemical evidence for direct targeting
of newly synthesized apical ABC transporters from the Golgi to the bile
canaliculus in vivo.
Newly Synthesized Canalicular ABC Transporters Are Directly
Targeted from the Golgi to the Hepatocyte Apical Domain in Rat
Liver*
*
This work was supported by Deutsche Forschungsgemeinschaft
Research Grant Ki 640 (to H. K.) and by NIDDK Grants DK35652 and 30DK34928 (Digestive Disease Center) from the National Institutes of
Health (to I. M. A.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6739; Fax: 617-636-0445; E-mail:
Irwin.Arias@Tufts.edu.
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