The
melanoma-associated
disialogangliosides 9(7)-O-acetyl-GD3 and 9(7)-O-acetyl-GD2 have been structurally well characterized. However, the compartmentalization and sequence of action of the biosynthetic activities responsible for synthesizing these molecules remain obscure. Here, we have studied the spatial and temporal interrelationships among the activities responsible for the later stages of
ganglioside biosynthesis and those for O-acetylation in cultured human
melanoma cells. First,
brefeldin A treatment was used to separate biosynthetic steps into compartments distal or proximal to the transport block imposed by the
drug. In keeping with prior reports, GM2/
GD2 synthase was consistently rendered inaccessible to its acceptors GM3 and GD3. In contrast, the effect on GD3 biosynthesis was cell line-specific. Synthesis of GD3 was nearly abrogated in two lines, while it accumulated in a third line. This indicates that the spatial organization of
ganglioside processing activities can vary even between similar cell lines. However, in all cell lines studied, the ratio of 9(7)-O-acetyl-GD3 to GD3 was not changed by
brefeldin A, indicating that the majority of
ganglioside O-acetyltransferase activity is co-localized with GD3 biosynthetic activity in the same Golgi subcompartment(s). As an alternative approach, Golgi-enriched fractions from
melanoma cells were incubated with radiolabeled and nonlabeled
nucleotide sugars or
acetyl-CoA. In these preparations, biosynthesis is dependent upon the co-localization of appropriate
sugar nucleotide transporters,
glycosyltransferases, and acceptors that are endogenously present within intact topologically correct compartments. Incubations with
CMP-Neu5Ac and
acetyl-CoA corroborated the results with
brefeldin A, co-localizing
ganglioside O-acetyltransferase activity in compartments where GD3 biosynthesis takes place. Analyses with
CMP-Neu5Ac and
UDP-GalNAc showed that GD2 and GD3 synthesis occur in partially overlapping compartments. Labeling with
acetyl-CoA and
UDP-GalNAc indicated that although labeled
acetate can be transferred from
acetyl-CoA directly to
GD2, ganglioside O-
acetyltransferase activity does not substantially overlap with the biosynthetic compartment(s) for GD2. Instead, O-acetyl-GD3 appears to be co-localized with the compartment of GD2 biosynthesis and serves as an acceptor for
GD2 synthase. Thus, both 9-O-acetyl-GD3 and GD2 can be precursors of 9-O-acetyl-GD2, but apparently in distinct compartments.