pH-dependent translocation of alpha-tocopherol transfer protein (alpha-TTP) between hepatic cytosol and late endosomes.
BACKGROUND
alpha-Tocopherol transfer protein (alpha-TTP), a member of the Sec14 protein family, plays an important role in transporting alpha-tocopherol, a major lipid-soluble anti-oxidant, in the cytosolic compartment of hepatocytes and is known as a product of the causative gene for familial isolated vitamin E deficiency. It has been shown that the secretion of hepatocyte alpha-tocopherol taken up with plasma lipoproteins is facilitated by alpha-TTP. To explore the mechanism of alpha-TTP mediated alpha-tocopherol secretion, we investigated drugs which may affect this secretion.


RESULTS
We found that, in a hepatocyte cell culture system, intracellular alpha-tocopherol transport is impaired by chloroquine, an agent known for its function of elevating the pH in acidic compartments. Under chloroquine treatment, the diffuse cytosolic distribution of alpha-TTP changes to a punctate pattern. Double-staining experiments with endocytosis markers revealed that alpha-TTP accumulates transiently on the cytoplasmic surface of late endosomal membranes. This phenomenon is specific for hepatoma cell lines or primarily cultured hepatocytes. Other members of the Sec14 family, such as cellular retinaldehyde-binding protein (CRALBP) and supernatant protein factor (SPF), do not show this accumulation. Furthermore, we elucidate that the obligatory amino acid sequence for this function is located between amino acids 21 and 50, upstream of the N-terminal end of the lipid-binding domain.


CONCLUSIONS
We hypothesize that a liver-specific target molecule for alpha-TTP exists on the late endosomal membrane surface. This transient binding may explain the mechanism of how alpha-tocopherol is transferred from late endosomes to cytosolic alpha-TTP.
Horiguchi
Masakuni
M
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
Arita
Makoto
M
Kaempf-Rotzoll
Daisy E
DE
Tsujimoto
Masafumi
M
Inoue
Keizo
K
Arai
Hiroyuki
H
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008660:Metabolism; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C017763:alpha-tocopherol transfer protein; D020126:Brefeldin A; D002738:Chloroquine; D003513:Cycloheximide; D015739:Nocodazole; D000077191:Wortmannin