Uncoupled packaging of targeting and cargo molecules during transport vesicle budding from the endoplasmic reticulum.
Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi 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 the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart. This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum. Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).
Yeung
T
T
Department of Molecular and Cell biology, Howard Hughes Medical Institute, University of California, Berkeley 94720, USA.
Barlowe
C
C
Schekman
R
R
D022181:COP-Coated Vesicles; D002458:Cell Fractionation; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006160:Guanosine Triphosphate / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins; D028861:Nuclear Pore Complex Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; C079620:SEC13 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006160:Guanosine Triphosphate; D003513:Cycloheximide; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae