Abstract
The trafficking of proteins along the first stage of the secretory pathway is mediated by small vesicles that bud from the endoplasmic reticulum (ER) and subsequently fuse with the cis-Golgi compartment. To follow vesicle formation, a differential centrifugation procedure is employed to separate the more rapidly sedimenting ER and Golgi membranes from the slowly sedimenting vesicles. Consumption is analyzed using a two-stage assay in which vesicles isolated by differential centrifugation during stage 1 are subsequently added to stage 2 (fusion) reactions containing acceptor Golgi membranes. Use a pipette to transfer the cells to 50-ml plastic tubes and then repeat the scraping procedure to ensure that all the cells are collected. Centrifuge at 720xg for 3 min and remove the supernatant by aspiration. Resuspend each cell pellet in 0.9 ml of homogenization buffer supplemented with PIC and homogenize by three complete passes through a 1-ml ball-bearing homogenizer. Remove the supernatants by aspiration, wash the pellets with 2 ml of transport buffer, and combine the membranes into two 1.5-ml microfuge tubes.
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