Abstract
Endocytosis is an essential process for the internalization of plasma membrane proteins, lipids and extracellular molecules into the cells. The mechanisms underlying endocytosis in plant cells involve several endosomal organelles whose origins and specific role needs still to be clarified. In this study we compare the internalization events of a GFP-tagged polygalacturonase-inhibiting protein of Phaseolus vulgaris (PGIP2-GFP) to that of a GFP-tagged subunit of cellulose synthase complex of Arabidopsis thaliana (secGFP-CesA6). Through the use of endocytic traffic chemical inhibitors (tyrphostin A23, salicylic acid, wortmannin, concanamycin A, Sortin 2, Endosidin 5 and BFA) it was evidenced that the two protein fusions were endocytosed through distinct endosomes with different mechanisms. PGIP2-GFP endocytosis is specifically sensitive to tyrphostin A23, salicylic acid and Sortin 2; furthermore, SYP51, a tSNARE with interfering effect on late steps of vacuolar traffic, affects its arrival in the central vacuole. SecGFP-CesA6, specifically sensitive to Endosidin 5, likely reaches the plasma membrane passing through the trans Golgi network (TGN), since the BFA treatment leads to the formation of BFA bodies, compatible with the aggregation of TGNs. BFA treatments determine the accumulation and tethering of the intracellular compartments labeled by both proteins, but PGIP2-GFP aggregated compartments overlap with those labeled by the endocytic dye FM4-64 while secGFP-CesA6 fills different compartments. Furthermore, secGFP-CesA6 co-localization with RFP-NIP1.1, marker of the direct ER-to-Vacuole traffic, in small compartments separated from ER suggests that secGFP-CesA6 is sorted through TGNs in which the direct contribution from the ER plays an important role. All together the data indicate the existence of a heterogeneous population of Golgi-independent TGNs.
Highlights
Endocytosis is a dynamic and complex cellular process required for the uptake of extracellular molecules, the regulation of plasma membrane protein and lipid turnover
Many plasma membrane proteins are present in monomeric form and are endocytosed via clathrin-mediated endocytosis but, in response to stress conditions, some of them form pre-dimers or dimers to subsequently assemble into clusters internalized via membrane microdomain-associated endocytic pathways
In tobacco cells it showed a patchy distribution on the plasma membrane (Supplementary Figures 2A–C) and accumulation in punctate compartments, likely Golgi bodies and small intracellular complexes closely associated to microtubules known as microtubule-associated cellulose synthase compartments (MASCs) (MicrotubuleAssociated cellulose Synthase Compartments) (Supplementary Figures 2D–F)
Summary
Endocytosis is a dynamic and complex cellular process required for the uptake of extracellular molecules, the regulation of plasma membrane protein and lipid turnover. It seems that microdomain-associated endocytosis occurs after protein clustering in responses to extracellular stimuli (Katritch et al, 2013). The cluster formation and clathrinindependent endocytosis provide an efficient mechanism to control the activity of membrane proteins in response to environmental changes (Fan et al, 2015)
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