An alternative angiosperm DGAT1 topology and potential motifs in the N-terminus

The highly variable cytoplasmic N-terminus of the plant diacylglycerol acyltransferase 1 (DGAT1) has been shown to have roles in oligomerization as well as allostery; however, the biological significance of the variation within this region is not understood. Comparing the coding sequences over the variable N-termini revealed the Poaceae DGAT1s contain relatively high GC compositional gradients as well as numerous direct and inverted repeats in this region. Using a variety of reciprocal chimeric DGAT1s from angiosperms we show that related N-termini had similar effects (positive or negative) on the accumulation of the recombinant protein in Saccharomyces cerevisiae. When expressed in Camelina sativa seeds the recombinant proteins of specific chimeras elevated total lipid content of the seeds as well as increased seed size. In addition, we combine N- and C-terminal as well as internal tags with high pH membrane reformation, protease protection and differential permeabilization. This led us to conclude the C-terminus is in the ER lumen; this contradicts earlier reports of the cytoplasmic location of plant DGAT1 C-termini.

Automated summary

The highly variable cytoplasmic N-terminus of plant DGAT1 plays important roles in oligomerization and allostery. However, the biological significance of variation within this region is not well understood. In this study, we found that Poaceae DGAT1s have high GC compositional gradients and numerous repeats in the variable N-terminus. Reciprocal chimeric DGAT1s in different angiosperms showed similar effects on protein accumulation in Saccharomyces cerevisiae. Furthermore, expression of specific chimeras in Camelina sativa seeds increased total lipid content and seed size. Our results also revealed the localization of the C-terminus in the ER lumen, contradicting earlier reports of its cytoplasmic location.