Thinking about p24 proteins and how transport vesicles select their cargo.

A ll cells are divided into distinct subcellular compartments, each with its own defining set of proteins. A fundamental problem in molecular cell biology is to understand how proteins that are synthesized on ribosomes in the cytoplasm reach their proper intracellular address. This process, usually called protein sorting or protein trafficking, is understood to involve information encoded in the protein sequence itself as well as the cellular machinery that decodes this information and delivers the protein to its correct location. Many of the sorting steps in a eukaryotic cell take place along the secretory pathway. At each step in the secretory pathway, carrier vesicles bud from one compartment and then fuse with the membrane of the next compartment allowing the transfer of membranes and proteins between organelles. To sort proteins, transport vesicles must be able to select the appropriate contents, accepting cargo proteins that should advance to the next compartment while excluding compartmental resident proteins that should remain in place. The companion article for this commentary concerns the function of the p24 family of vesicle proteins, which are thought to play an important part in cargo selection (1). Our current understanding of how vesicles select specific cargo molecules can be traced back to studies of how extracellular proteins are transported into the cell interior by endocytosis (2). A schematic diagram of the molecular associations that drive endocytic vesicle assembly and cargo loading into these vesicles is shown in Fig. 1. The individual steps (reviewed in ref. 3) are as follows: clathrin and adaptor protein complexes form the coats of endocytic vesicles, and as these coat proteins polymerize onto the plasma membrane, the resulting deformation of the membrane into a nascent vesicle produces a structure known as a coated pit. Because membrane receptor proteins have short cytoplasmic sequences with affinity for coat proteins, the polymerized coat acts as affinity matrix to cluster receptor molecules into the coated pits. The extracellular domains of receptors, such as the low density lipoprotein receptor, in their turn bind to their corresponding ligands to collect them into the forming vesicle. Further polymerization of the coat sets the curvature of the membrane into a spherical shell, and finally, with the aid of accessory proteins such as dynamin, a completed vesicle is pinched off from the membrane. This process of coat assembly coupled to the selection of cargo by membrane receptors seems to be quite general, because cargo receptors mediate protein sorting in the trans-Golgi (4, 5) and retrieval of mistargeted proteins from the Golgi to the endoplasmic reticulum (ER; ref. 6). For ER to Golgi transport, the first vesicle trafficking step in the secretory pathway, cargo receptors have not been identified; however, some type of cargo selection mechanism is expected, because cargo is concentrated into ER-derived vesicles (7, 8), whereas organelle resident proteins are excluded (9). The p24 proteins are a conserved family of small integral membrane proteins found in eukaryotes from yeast to mammals. These proteins were first identified as abundant constituents of the COPI and COPII vesicles that operate in the early secretory pathway (10–12). (COPI vesicles carry proteins between the cisternae of the Golgi complex and from the Golgi to the ER, whereas COPII vesicles carry proteins from the ER to the Golgi.) Because of their abundance, their conservation through evolution, and the fact that they shuttle between the ER and Golgi compartments in transport vesicles, the p24 proteins are thought to be fundamental constituents of vesicles, perhaps acting as cargo receptors. One approach to define the physiological function of the p24 proteins has been to mutate the corresponding genes and then to look for associated defects in vesicular trafficking. Such genetic tests have been applied to the yeast Saccharomyces cerevisiae, but they have not yielded simple answers. Yeast strains carrying mutations in p24 genes grow normally, and overt defects in either COPI or COPII vesicle functions are not seen (11, 13). A confounding problem in the interpretation of these experiments is the redundancy of p24 genes. The S. cerevisiae genome carries eight p24 homologs, and if these proteins can substitute even partly for one another, one cannot be sure of the consequences of elimination of the p24 function until all eight homologs have been knocked out. This octuple mutant has now been constructed and has no detectable defect in the rate of ER to Golgi transport (as measured by the kinetics of carboxypeptidase Y export from the ER) or in the rate of transport from the Golgi back to the ER (as measured by the ability of a reporter protein bearing a KKXX retrieval sequence to be recycled; ref. 1). These results show that the p24 proteins are not essential in yeast for the function of either COPI or COPII vesicles. Although they do not seem to cause a pronounced defect in vesicle trafficking, p24 gene mutations in yeast do cause subtle alterations in the secretory pathway, which may provide important clues as to their function. The EMP24 gene was the first of the yeast p24 proteins to be identified. Morphological characterization of a deletion of EMP24 revealed an approximately 2-fold reduction in the production of small vesicles, although the identity of the affected vesicle class as either COPI or COPII could not be established (10). Parallel experiments examining the kinetics of protein trafficking in the early secretory pathway revealed that deletion of either EMP24 or ERV25 (a second p24 homolog) has no observable effect on carboxypeptidase Y export and slows export from the ER of invertase and proteins linked to glycosylphosphatidylinositol (GPI) anchors (11, 13). This difference in the rates of export for different protein cargo molecules suggests that EMP24 and ERV25 might encode cargo receptors for inver-