The mammalian Golgi apparatus plays a central role in the secretory pathway and in sphingolipid synthesis. The complex architecture of the mammalian Golgi ribbon with ER-trans Golgi contact sites is thought to facilitate delivery of ceramide from the ER for synthesis of SM at the trans
]. We previously showed that certain Golgi morphologies (induced by drugs that perturb Golgi structure) promote efficient ceramide trafficking and SM synthesis, while others do not [23
]. Efficient SM synthesis correlated with CERT localization at Golgi membranes. A recent study also found a dramatic decrease in SM synthesis in cells deficient in conserved oligomeric Golgi complex-2 (Cog2), a component of a retrograde trafficking complex that is required for proper localization of certain glycosyltransferases [27
]. The morphology of the Golgi complex in Cog2 null cells is disrupted, which may interfere with proper CERT localization. Thus, Golgi architecture is important for efficient SM synthesis. Here, we showed that induction of proapoptotic pathways by TNFα and anisomycin results in disassembly of the Golgi complex, probably due to cleavage of Golgi structural proteins including golgin-160, giantin, p115 and GM130 by activated caspases [4
]. In addition, to Golgi disassembly, treatment with proapoptotic drugs resulted in a decrease of CERT at the Golgi region, caspase cleavage of CERT, and a large reduction in SM synthesis.
CERT was cleaved by caspases-2, 3, and 9 in vitro
, and we mapped the caspase-2 cleavage site to D213
in the sequence TTRSD. Although this sequence is not predicted to be a good caspase-2 target [37
], the best-characterized caspase-2 cleavage sequence (GESPD in golgin-160) does not fit the prediction either. The Kcat
for cleavage of CERT by caspase-2 was 4.02 × 103
, and the Kcat
for cleavage of golgin-160 was 4.38 × 103
. Thus, CERT is almost as good a substrate for caspase-2 as golgin-160. The Kcat
values measured by us for cleavage of golgin-160 by caspase-2 was less than the Kcat
value (33 × 103
) reported previously [4
]. This discrepancy could result from our use of the N-terminal portion instead of the full-length golgin-160 substrate, or a different preparation of recombinant caspase-2. Although we did not map the sites of cleavage for caspases-3 and -9, we found that CERT-D213
A was still cleaved in cells treated with proapoptotic drugs at sites downstream of D213
. Thus, even though caspase-2 may contribute to inactivation of CERT, other caspases can also perform this role if the caspase-2 site is absent or if activation of procaspase-2 does not occur.
Interestingly, although anisomycin and TNFα treatments resulted in about 45% and 26% decrease in full-length endogenous CERT, newly synthesized SM levels decreased by about 71% and 82%, respectively. Thus, it is likely that Golgi disassembly and CERT mislocalization also contribute to the impairment of SM synthesis, thereby causing a greater reduction in newly synthesized SM than would be expected with just CERT cleavage alone.
Changes in Golgi morphology during early times (1h) of proapoptotic treatment were subtle, and we observed CERT mislocalization, reduction in full-length protein, and impairment of SM synthesis only after 4h of proapoptotic treatment when Golgi alterations were more significant. Activation of initiator caspases and cleavage of a subset of Golgi substrates at early times may lead to small changes in Golgi organization. Subsequent activation of effector caspases may then result in gross morphological changes of the Golgi apparatus, including generation of dispersed Golgi membranes seen at later times. It is also possible that gross morphological changes at later time points correlate with dysregulation of cellular ceramide levels, thus amplifying the proapoptotic signaling pathway. Ceramide is also thought to play a role in maintenance of structural integrity of the ER and the Golgi [38
], as well as in apoptotic signaling pathways [16
]. Cells experiencing mild stress may activate survival responses (e.g. the unfolded protein response). However, cells exposed to prolonged or severe stress may activate caspases and commit to the apoptotic pathway [39
]. One route for this could be inhibition of SM synthesis and an increase in the ceramide pool. However, at 4h of TNFα and anisomycin treatments we did not observe a significant increase in newly synthesized ceramide. Since we were only analyzing de novo
synthesis of ceramide during the proapoptotic treatments, we cannot rule out generation of unlabeled ceramide from SM, sphingosine and other sphingolipids in different regions of the cell. Previous studies have indicated that TNFα treatment in particular, is associated with a rapid increase in ceramide levels at the plasma membrane by the activation of sphingomyelinase [40
]. Thus, understanding how cells sense ceramide levels at the ER and the Golgi [12
], and integrate proapoptotic signaling is imperative. This would help us examine the possibility that the Golgi apparatus acts as a platform to integrate proapoptotic and sphingolipid signaling pathways via caspases and CERT.
Inactivation of CERT and inhibition of SM synthesis results in decreased utilization of substrates ceramide and phosphatidylcholine (PC). While the consequence of increased ceramide levels in cells has been studied (discussed above), it is not clear how cells may handle PC when SM synthesis is inhibited during proapoptotic treatments [42
]. When SM is synthesized in the trans
Golgi from substrates ceramide and PC, diacylglycerol (DAG) is produced [43
]. DAG at Golgi membranes serves to recruit protein kinase D (PKD), which regulates vesicle production and thus plays a role in modulating cargo trafficking through the organelle [45
]. If SM synthesis decreases due to inactivation of CERT, DAG and consequently PKD recruitment to Golgi membranes would also presumably decrease, suggesting that CERT may function indirectly in cargo trafficking through the Golgi apparatus [48
]. Additionally, altering SM synthesis in the Golgi and accordingly SM levels along the secretory pathway may also alter the SM/cholesterol ratio [49
], thereby affecting partitioning and trafficking of lipids and proteins to their target destination (e.g. proteins targeted to lipid rafts in the plasma membrane). Thus, CERT may play an important role in regulating both lipids and proteins when cells are under severe stress.
In this study we have shown that both Golgi disassembly and inactivation of CERT contribute to decreased SM synthesis during proapoptotic stress. A more comprehensive approach for determining the order in which apoptotic signaling, ceramide accumulation, and Golgi disassembly occur will be required to understand how the Golgi apparatus contributes to cellular stress sensing and integration of apoptosis signaling.