Sphingolipids mediate polar sorting of PIN2 through phosphoinositide consumption at the trans-Golgi network

Abstract

The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting.

StREM1.3 REMORIN Protein Plays an Agonistic Role in Potyvirus Cell-to-Cell Movement in N. benthamiana

“…The colocalisation between the YFP signal reconstituted in BiFC (nYFP–REM and CI–cYFP) and aniline blue labelling at the PD level was quantified using the centroid object-based method using the 3D objects counter plugin of ImageJ software [ 47 ]. The resolution limit of each image was determined with a numerical aperture of 1.4 for x63 oil objective and the shorter emission maximum wavelength of the fluorophores.…”

Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments

“…While the intensity of the mCIT-3xPH FAPP1 at the PM decreased already after 15 min of wortmannin treatment and became even more weaker at 90 min of treatment, the fluorescence intensity at intracellular dots was not modified even after 90 min treatment (Figure 4b,c). We previously found that metazachlor treatment causes the accumulation of PI4P at TGN [38], this accumulation was not modified by additional wortmannin treatment (Figure 4c). These results show that wortmannin impacts PI4P quantity at the PM but not at intracellular compartments.…”

“…In animal cells, lipid exchange through membrane contact sites have been evidenced especially in the case of PI4P/sterol exchange [45,46]. It is unlikely that this sterol-exchange mechanism is controlled by VLCFAs in plant cells as reducing the acyl-chain length by metazachlor does not alter sterol content in microsomal or TGN-purified fraction [10,38]. We could not test this hypothesis further during the course of this study due to the technical challenge of measuring fluorescence intensity variations specifically during TGN/PI3P-positive MVB associations.…”

Phosphatidylinositol 4‐phosphate: a key determinant of plasma membrane identity and function in plants

“…However, the two binding sites are distinct, so it is possible to mutate the ARF1 binding site while leaving the PI4P interacting domain fully functional (He et al ., 2011). Such a mutant strictly localizes to the plasma membrane in plants (Simon et al ., 2016; Ito et al ., 2021). By contrast, mutants still able to interact with ARF1 but not PI4P localize to the TGN and not the plasma membrane (Simon et al ., 2016).…”

“…This phenotype is even stronger in sac6sac7 and sac7sac8 double mutants, SAC6 and SAC8 being closely related to the SAC7 protein (Dubois & Jaillais, 2020; Song et al ., 2021). This balance is also maintained by the activity of a phosphoinositide‐specific phospholipase C (PI‐PLC) at the TGN (Ito et al ., 2021). Chemical inhibition of PI‐PLCs increases the amount of PI4P sensors in the TGN.…”