Polarized Sphingolipid Transport from the Subapical Compartment: Evidence for Distinct Sphingolipid Domains

Department of Physiological Chemistry, Faculty of Medical Sciences, University of Groningen, 9713 AV Groningen, The Netherlands.
Molecular Biology of the Cell (Impact Factor: 4.47). 11/1999; 10(10):3449-61. DOI: 10.1091/mbc.10.10.3449
Source: PubMed


In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR-dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of "raft" formation and apical targeting is discussed.

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    • "Bile canalicular lumens were first identified with phase contrast and scored as NBD positive or negative with epifluorescence. Figure 6, A–D, shows that the lipid analogue reached the apical surface domain in untreated or OSM-treated cells expressing the AKAP-IS peptide, similarly as previously shown in control HepG2 cells (Zegers and Hoekstra, 1997; van IJzendoorn and Hoekstra, 1999, 2000; van der Wouden et al., 2002) and consistent with previous results (Wojtal et al., 2006). Basolaterally derived NBD-sphingomyelin reached 74 and 89% of the bile canalicular lumens after a chase of 15 and 30 min, respectively, in parental cells. "
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    • "Because the data strongly indicate that the establishment of HepG2 cell polarity is critically dependent on the level of sphinganine, we next investigated whether dihydroceramide synthase, the predominant enzyme in sphinganine turnover , functions as a target for cellular signals that contribute to polarity development. For this, we took into account our previous observation that the stable cAMP analog dbcAMP, via activation of PKA, stimulates HepG2 cell polarity development (Zegers and Hoekstra, 1997; van IJzendoorn and Hoekstra, 1999a, 2000). Importantly, quantification of sphinganine from cell extracts by ESI-MS/MS revealed that the cellular sphinganine level was reduced with ϳ63% in dbcAMP-treated cells, compared with nontreated cells (Table 2), which is in agreement with the observation that reduced sphinganine levels promote polarity development. "
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