Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes.
BACKGROUND
In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level.


RESULTS
Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations.


CONCLUSIONS
Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.
Grebe
Markus
M
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. markus.grebe@genfys.slu.se
Xu
Jian
J
Möbius
Wiebke
W
Ueda
Takashi
T
Nakano
Akihiko
A
Geuze
Hans J
HJ
Rook
Martin B
MB
Scheres
Ben
B
D000199:Actins / Q000378:metabolism*; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology; D018744:DNA, Plant / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D029681:Arabidopsis Proteins; D020126:Brefeldin A; D018744:DNA, Plant; D020558:GTP Phosphohydrolases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013261:Sterols; D020691:rab GTP-Binding Proteins; C117013:AT5G57090 protein, Arabidopsis; C436706:Ara6 protein, Arabidopsis