Abstract
Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.
Highlights
Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer)
Our findings suggest that the heteromeric complex of Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) is involved in the regulation of Cer metabolism, which may be one of the reasons why Cer transport protein (CERT)-transported Cer is used for SM synthesis
As expected on the basis of previous reports [3, 13], confocal microscopy showed that SMS1 strongly co-localized with GCS and the trans-Golgi network marker, p230, whereas SMS2 only partially co-localized with GCS and p230 (Fig. 1A)
Summary
Before investigating the complex formation of SMS1 with GCS, the co-localization of both enzymes was examined. The decreases in [14C]stearic acid-derived GlcCer by HPA-12 treatment in HEK293T and SMS1 KO cells might be due to a secondary effect of CERT inhibition (Fig. 9E), [14C]stearic acid-derived Cer was not affected by HPA-12 (Fig. 9F) To confirm their contribution to the SMS1–GCS activities, another substrate, [3H]sphingosine, was employed for metabolic labeling, which bypasses de novo synthesis of sphingolipid bases in the labeling of Cer [12, 30, 31]. HPA-12 treatment in SMS1 KO cells decreased CERT-dependent GlcCer synthesis in trans-Golgi, whereas there was an increase in GlcCer synthesis from the overflow of Cer in cis-Golgi, and as a result, overall GlcCer levels were not substantially affected (Fig. S2B) These results suggested that CERT-transported Cer in transGolgi is partly regulated by the SMS1–GCS heteromeric complex in HEK293T cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
