The Sorting of Membrane Proteins During the Formation of Er-Derived Transport Vesicles

Abstract

Proteins enter the transport pathway of eukaryotic cells when they are translocated into or across the membrane of the ER. Once in the transport pathway, proteins can be secreted or targeted to the Golgi cisternae, lysosomes, or the plasma membrane. Repeated cycles of vesicle budding and fusion move proteins through the transport pathway. In the yeast Saccharomyces cerevisiae, a combined genetic and biochemical approach has identified many of the components required for the vesicular transport of proteins from the ER to the Golgi (Salama et al. , 1993; Barlowe et al. , 1994). The formation of ER-derived vesicles from ER membranes in vitro has now been observed, and this assay requires the addition of cytosol or a specific set of proteins (Sarlp, Sec23p complex, and Secl3p complex). The vesicles formed in this assay will fuse with Golgi membranes in vitro. Additionally, this reaction retains the selectivity observed in vivo. Specifically, ER resident proteins such as Sec61p and Secl2p are not included in these vesicles. Despite this progress, little is known about how protein sorting occurs during the process of vesicular budding from ER membranes. Several amino acid sequences have been found that are responsible for the ER-localization of proteins. For example, the C-terminal addition of the sequence HDEL to soluble proteins or KKXX to membrane-bound proteins leads to ER localization. These sequences do not however, appear to lead to the exclusion of proteins from ER-derived transport vesicles. Instead, proteins containing such sequences are retrieved from early Golgi compartments after they have been released from the ER. To date, no sequences that affect the sorting of proteins into ER derived transport vesicles have been identified. As a first step towards identifying such signals, we have constructed fusions between two type II membrane proteins that display different sorting properties. One of these proteins, Sec22p is efficiently packaged into ER-derived transport vesicles and the other, Secl2p, is not. Our results with these fusions indicate that sorting information is present on the cytoplasmic domain of one or both of these proteins.