UNCOUPLED PACKAGING OF TARGETING AND CARGO MOLECULES DURING TRANSPORTVESICLE BUDDING FROM THE ENDOPLASMIC-RETICULUM

Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Gels apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins. In order to examine th e importance of cargo in this packaging reaction, we developed an in v itro assay that quantifies vesicle formation based on segregation of t argeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of target ing molecule-containing vesicles in a fashion indistinguishable from i ts normal counterpart. This observation implies that packaging of carg o proteins may be uncoupled from the recruitment of targeting molecule s during vesicle budding from the endoplasmic reticulum. Using the sam e assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).