Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis.
The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.
Department of Molecular Cellular and Developmental Biology, University of Colorado-Boulder, Boulder, CO 80309, USA. bkang@ufl.edu
Byung-Ho
BH
Kang
Erik
E
Nielsen
Mary Lai
ML
Preuss
David
D
Mastronarde
L Andrew
LA
Staehelin
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D048749:Cytokinesis / Q000502:physiology; D055032:Electron Microscope Tomography; D055786:Gene Knockout Techniques; D007150:Immunohistochemistry; D009154:Mutation; D018517:Plant Roots / Q000201:enzymology / Q000648:ultrastructure; D011134:Polysaccharides / Q000032:analysis; D022142:Secretory Vesicles / Q000648:ultrastructure; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000737:chemistry / Q000648:ultrastructure
D029681:Arabidopsis Proteins; D011134:Polysaccharides; D019870:1-Phosphatidylinositol 4-Kinase; D020695:rab4 GTP-Binding Proteins