Membrane environment-dependent relocation of phloem-loading sucrose transporters.

Plasma membrane transporters play an essential role in plant development and all physiological processes. Their activity is tightly controlled by a variety of mechanisms, including the rate of transporter endocytosis. There is evidence that the local membrane environment, in particular the sterol content, influences transporter endocytosis. Here, we analyzed the sucrose transporters StSUT1 (potato) and AtSUC2 (Arabidopsis) transiently expressed in Nicotiana benthamiana epidermal cells. Using live-cell imaging of fluorescent protein fusions combined with extracellular quenching by Trypan Blue, we quantitatively distinguished plasma membrane-localized from internal transporter pools. Pharmacological perturbation of sterols and sphingolipids with methyl-β-cyclodextrin, fenpropimorph and myriocin reduced sucrose transporter abundance at the plasma membrane and decreased uptake of the fluorescent sucrose analogue esculin. In Arabidopsis leaves, methyl-β-cyclodextrin treatment reduced endogenous AtSUC2 protein levels, confirming sterol-dependent regulation in planta. Single-molecule localization microscopy of AtSUC2-reporter constructs revealed its enrichment in ∼50 nm plasma membrane domains, with the localization becoming more homogeneous upon sterol depletion. Together, our results demonstrate that plasma membrane lipid composition modulates sucrose transporter localization and abundance, with likely relevance for the regulation of phloem loading and other membrane proteins.