Abstract
Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56-amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and >400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by expression of wild-type ssSPTa or ssSPTb under control of the ssSPTa promoter, indicating ssSPTa and ssSPTb functional redundancy. SPT activity and sensitivity to the PCD-inducing mycotoxin fumonisin B1 (FB1) were increased by ssSPTa overexpression. Conversely, SPT activity and FB1 sensitivity were reduced in ssSPTa RNA interference lines. These results demonstrate that ssSPTs are essential for male gametophytes, are important for FB1 sensitivity, and limit sphingolipid synthesis in planta.
Highlights
Sphingolipids are essential components of eukaryotic cells with diverse roles in membrane structure and function and mediation of basic cellular processes, such as programmed cell death (PCD) (Brodersen et al, 2002; Liang et al, 2003; Alden et al, 2011; Markham et al, 2013)
Expression of LCB1-FLAG with the Myc-LCB2 subunits failed to complement the long-chain-base auxotrophy of the yeast mutant, but coexpression of either HAssSPTa or HA-ssSPTb resulted in robust growth, even at 37°C where the requirement for Serine palmitoyltransferase (SPT) activity is relatively high (Figure 2A)
Immunoprecipitation of the LCB1-FLAG subunit resulted in copurification of the HA-subunits of SPT (ssSPT) as well as the Myc-LCB2 subunits (Figure 2B, left panel)
Summary
Sphingolipids are essential components of eukaryotic cells with diverse roles in membrane structure and function and mediation of basic cellular processes, such as programmed cell death (PCD) (Brodersen et al, 2002; Liang et al, 2003; Alden et al, 2011; Markham et al, 2013). Sphingolipids contribute to the structural integrity of raft-like domains in the plasma membrane that are important for cell surface activities, including cell wall synthesis and degradation, signaling, and trafficking (Mongrand et al, 2004; Borner et al, 2005; Melser et al, 2011). Beyond their functions in membranes, Maintenance of sphingolipid homeostasis is critical for all eukaryotic cells. Sphingolipid homeostasis is generally believed to be mediated by regulation of serine palmitoyltransferase (SPT), the first enzyme in long-chain-base biosynthesis that catalyzes the condensation of Ser with typically palmitoyl (16:0)- or stearoyl (18:0)-coenzyme A (CoA)
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