In situ localization and in vitro induction of plant COPI-coated vesicles.
Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.
Pimpl
P
P
Department of Structural Cell Physiology, Albrecht-von-Haller Institute of Plant Sciences, University of Göttingen, Germany.
Movafeghi
A
A
Coughlan
S
S
Denecke
J
J
Hillmer
S
S
Robinson
D G
DG
D001483:Base Sequence; D020126:Brefeldin A / Q000494:pharmacology; D003429:Cross Reactions; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007106:Immune Sera; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D021381:Protein Transport
D017931:DNA Primers; D007106:Immune Sera; D010940:Plant Proteins; D020126:Brefeldin A