Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.
Bottom Line: Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins?We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other.Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.
Affiliation: Cell and Developmental Biology Programme, Centre for Genomic Regulation, 08003 Barcelona, Spain Universitat Pompeu Fabra, 08002 Barcelona, Spain.Show MeSH
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Mentions: As the localization of Golgi glycosylation enzymes is affected by treatment with d-cer-C6 (Fig. 1), we investigated whether protein glycosylation was also affected. TGN46 is a transmembrane protein that is localized to the TGN and cycles between the TGN and the plasma membrane (Banting and Ponnambalam, 1997). The core protein of TGN46 has a molecular mass of 46 kD, but as a result of various glycosylations occurring at the ER and the Golgi complex, the mature protein has an apparent molecular mass of ∼110 kD (Prescott et al., 1997). HeLa cells were treated with increasing concentrations of d-cer-C6 for 4 h, after which the apparent size of TGN46 was examined by Western blotting. Whereas in cells treated with ethanol or l-cer-C6, TGN46 mainly appears as a fully processed 110-kD band, a smaller form of ∼80 kD becomes the main form upon treatments with increasing concentrations of d-cer-C6, indicating an incomplete processing of the protein (Fig. 4 A). This 80-kD form of TGN46 corresponds to a newly synthesized pool because this band was not evident in cells treated with d-cer-C6 in the presence of cycloheximide to stop new protein synthesis (Fig. S1).
Affiliation: Cell and Developmental Biology Programme, Centre for Genomic Regulation, 08003 Barcelona, Spain Universitat Pompeu Fabra, 08002 Barcelona, Spain.