Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80exit the Golgi apparatus of developing pea cotyledons in different transport vesicles

In the parenchyma cells of developing legume cotyledons, storage proteins a re deposited in a special type of vacuole, known as the protein storage vac uole (PSV). Storage proteins are synthesized at the endoplasmic reticulum a nd pass through the Golgi apparatus. In contrast to lysosomal acid hydrolas es, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-d ense vesicles rather than in clathrin-coated vesicles. By combining isopycn ic and rate zonal sucrose density gradient centrifugation with phase partit ioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage p roteins. In dense vesicles, prolegumin occurred in a more aggregated form t han it did in the endoprasmic reticulum. The putative vacuolar sorting rece ptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly redu ced in the dense vesicle fraction. This result was confirmed by quantitativ e immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of t he dense vesicles could not be detected. In contrast, 90% of the dense vesi cles were labeled with antibodies raised against alpha-TIP (for tonoplast i ntrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and alpha- TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.