When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.

The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.

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Membrane proteins are transported in vesicles or tubules that carry their cargo from one subcellular site to another. A crucial step in protein targeting is thought to occur during formation of these vesicles; peptide signals within the protein's sequence interact with soluble factors that govern cargo selection into nascent vesicles. The use of the GFP tag has revolutionized imaging of transport vesicles in live cells, and the development of GFP color variants that can be used in combination (such as CFP and YFP) makes it possible to watch two different proteins travel at the same time in the same cell [1,2]. We have applied dual-color, CFP,YFP imaging of transport carriers to a key issue in nerve cell biology, and an issue of importance to cell biology in general: characterizing the vesicle populations that deliver proteins to distinct domains on the plasma membrane.Neurons in culture are particularly well suited for live cell imaging studies of polarized cells. First, they are relatively flat, so capturing movies of transport carriers in axons and dendrites is possible. In addition, vesicles moving back and forth in axons and dendrites essentially travel along a single axis, which makes them easy to follow and analyze. To examine the specific packaging of membrane proteins into transport carriers, we used GFP color variants to tag dendritically and axonally polarized proteins. To determine if two markers were transported together, we imaged cells expressing CFP and YFP pairs, taking sequential 800 msec exposures in the two channels over a time period of 48 seconds and analyzed vesicle movements in such movies using the kymograph function in Metamorph. When displayed as a kymograph, moving organelles are represented as diagonal lines whose slopes correspond to the velocity of movement; stationary structures appear as horizontal lines. As a control, we made two-color movies of cells co-transfected with TfR-CFP and TfR-YFP (Figure 1), which exhibited a high degree of overlap (about 90%, see Table 1).Using this approach, we found that representative axonal (NgCAM) and dendritic (TfR, LDLR) markers were present in separate carrier populations, while the two dendritic markers were found in overlapping carrier populations. In addition, the sorting of LDLR was altered upon mutation of critical residues in its sorting signal, shifting LDLR out of TfR-containing dendritic transport vesicles and into an NgCAM-positive vesicle population (Table 1). References[1] P. T. Keller et al. Nature Cell Biology 3, (2001) 140.[2] This work was supported by NIH Grants MH66179 and NS17112. We thank B. Smoody for preparing hippocampal cultures and J. Luisi Harp for assistance with molecular biology.