In situ localization and in vitro induction of plant COPI-coated vesicles

Coat protein (COP)-coated vesicles have been shown to mediate protein trans port through early steps of the secretory pathway in yeast and mammalian ce lls. Here, we attempt to elucidate their role in vesicular trafficking of p lant cells, using a combined biochemical and ultrastructural approach. Immu nogold labeling of cryosections revealed that COPI proteins are localized t o microvesicles surrounding or budding from the Golgi apparatus. COPI-coate d buds primarily reside on the cia-face of the Golgi stack. In addition, CO PI and Arf1p show predominant labeling of the cis-Golgi stack, gradually di minishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induct ion experiments demonstrated that Arf1p as well as coatomer could be recrui ted from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi me mbranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underli ning 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 c entrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha -amyl ase-HDEL yielded a COPI-coated vesicle fraction that contained ol-amylase a s well as calreticulin.