A VAST staging area for regulatory proteins.

T he vesicle trafficking system has long been known to influence signal transduction through its role in biogenesis and degradation of membrane receptors (1). There are also well known examples of vesicleassociated regulatory proteins, such as Ire1 and SREBP, that control responses closely tied to vesicle properties or function (2, 3). But a new paradigm is emerging, as illustrated by a study in this issue of PNAS (4), in which vesicleassociated signal transduction (VAST) may not be related to trafficking of a transmembrane receptor. Rather, vesicles serve as staging areas for regulatory proteins, facilitating interactions that ultimately lead to activation or effector interaction (Fig. 1). The report by Puria et al. in this issue of PNAS (4) focuses on yeast Gln3, a transcription factor that is activated by TORC1 kinase inhibition (5) or by growth on poor nitrogen sources (6). A set of vesicle trafficking proteins were recently implicated in TORC1 kinase function (7). The new study shows that these vesicle trafficking proteins—class C and D Vps proteins—are required for Gln3 to translocate into the nucleus and activate transcription specifically in response to the nitrogen signal. These particular Vps proteins were known to be required for Golgi-to-endosome vesicle trafficking. Thus the genetic analysis indicates that Golgi-to-endosome vesicle trafficking is required to relay a specific Gln3-activating signal. On their own, these observations are consistent with indirect effects of the Vps proteins, such as the possibility that they are required for biogenesis of a plasma membrane receptor. However, the analysis of Gln3 subcellular localization points to a more direct relationship. In wild-type cells, inactive Gln3 is found in both cytoplasmic and membrane fractions. However, in mutants lacking either of two Vps proteins, Gln3 is almost entirely membrane associated. Moreover, sucrose gradient sedimentation analysis and immunofluorescence microscopy confirm that, in wild-type cells, a fraction of inactive Gln3 is associated with the Golgi and endosome marker protein Vps10. The finding that inactive Gln3 is associated with Golgi or endosomal membranes, and that Golgi-to-endosome trafficking defects impair Gln3 activation, together argue that Gln3 trafficking from Golgi to endosome is required for Gln3 activation. What tethers Gln3 to membranes? Two Gln3 regulators, TORC1 kinase and Gln3 inhibitor Ure2, are both associated with membranes as well. However, deletion derivatives of Gln3 that lack the TORC1-binding or Ure2binding regions remain membrane associated. Perhaps either interaction is sufficient for Gln3 membrane binding, or an as-yet-unknown binding partner may govern localization. What is the mechanistic consequence of Gln3-membrane association? Puria et al. (4) speculate that Gln3 (or the Gln3– Ure2 complex) may be conveyed to the endosome-associated TORC1 complex by Golgi-derived vesicles, a crucial step for Gln3 nuclear translocation. What is the significance of this vesicle-associated regulatory interaction? Binding of each protein to the same membrane surface should stabilize otherwise weak protein–protein interactions by limiting diffusion. But in addition, Puria et al. (4) point to growing evidence that the protein secretory pathway has a more direct role in nutrient sensing, as manifested through nutrientdependent sorting of the nitrogenregulated permease Gap1. Thus association of Gln3 with the secretory pathway may be a critical step for transduction of a nitrogen limitation signal. Transient endosome association has emerged as a pivotal regulatory event in two other signaling pathways. One is the Saccharomyces cerevisiae matingresponse pathway (8). A heterotrimeric G protein relays the activating signal, and both the G subunit (called Gpa1) and, independently, the G complex interact with effectors. Slessareva et al. (9) recently identified the Gpa1 effector: endosome-associated Vps34, the lone yeast phosphatidylinositol 3-kinase. Inactive Gpa1 is found predominantly at the plasma membrane, but Slessareva et al. found that an activated mutant Gpa1 is associated with an endosomal compartment along with Vps34 (Fig. 1B). They propose that activation of Gpa1 causes its dissociation from plasma membranebound G , permitting its translocation to endosomal surfaces where it stimulates Vps34. The other endosome-associated signaling pathway is the fungal Rim101/ PacC pathway, which is activated by growth in nonacidic environments (10). Activation results in proteolytic removal