Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41°C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41°C even in the presence of LMB. The temper- ature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.Key regulators of the heat stress (HS) response are the HS transcription factors (Hsfs), which belong to a family of proteins conserved throughout the eukaryotic kingdom (24,26,35,46). Hsfs have a modular structure with an N-terminal DNAbinding domain characterized by a helix-turn-helix motif, an adjacent domain with heptad hydrophobic repeats (HR-A/B) involved in oligomerization, a cluster of basic amino acid residues essential for nuclear import (the nuclear localization signal, or NLS) and a C-terminal activation domain (Fig. 1).The high degree of structural and functional conservation of Hsfs was documented repeatedly by using heterologous systems for Hsf expression in combination with appropriate reporter assays. Thus, Drosophila melanogaster and human Hsfs were tested in plant cells, Xenopus oocytes, and Saccharomyces cerevisiae (5,6,22,43,50), and plant Hsfs were tested in yeast, Drosophila, and human cells (4,5,8,17). Using yeast strains with disruption of the endogenous Hsf1 gene, it was shown that many of these heterologous Hsfs were able to replace the yeast Hsf1 in most of its functions, i.e., in Hsf-dependent reporter assays, in the survival function both at 25 and 37°C, and in the generation of the thermotolerant state (5,12,22,48).In plants, the Hsf system is more complex than in any other organisms investigated so far (26,28,35). (i) Besides the constitutively expressed members of the Hsf family, many Hsfs themselves are HS-i...

Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuroendocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of smaUl vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5Ј-[␥-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface. INTRODUCTIONProteins internalized via receptor-mediated endocytosis are rapidly transported via clathrin-coated vesicles to endosomal structures located in the periphery of the cell known as early or sorting endosomes (Gruenberg and Maxfield, 1995;Mellman, 1996;Clague, 1998). From there, ligands and their cognate receptor as well as fluid material are delivered via late endosomes to lysosomes where they are degraded. Some of the receptors, including the transferrin (Tf) receptor (TfR), are recycled back to the cell surface from the peripheral endosomes, either directly or indirectly, via pericentriolar recycling endosomes.One of the unsolved issues is the mechanism by which those plasma membrane proteins that are not degraded are transported out of the sorting endosomes. It is not clear whether the transport vehicles are tubules or vesicles. It is also not known in molecular terms how recycling membrane proteins are sorted from each other in the sorting endosome. For example, there is evidence that membrane proteins are transported to the recycling endosomes by default, following the bulk flow of lipids (Mayor et al., 1993). On the other hand, there has been circumstantial evidence to support a role for coated vesicles in the recycling of these proteins from endosomes. A number of coat proteins copurify with fractions enriched in early endosomes (Whitney et al., 1995).Electron microscopic studies also demonstrate budding profiles on endosomes, many containing clathrin and other coat proteins (Stoorvogel et al., 1996;Futter et al., 1998). Endo- somes from specialized neuroendocrine PC12 cells have also been shown to give rise to a subset of synap...

Abstract. We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221. Although plgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.p LASMA membrane proteins that normally reside in axonal domains of neurons are selectively targeted to apical domains when expressed in epithelial cells, suggesting an overlap between epithelial and neuronal-targeting mechanisms (Dotti et al., 1991;Powell et al., 1991;Pietrini et al., 1994). The overlap may include endocytotic structures as well, since apical and axonal early endosomes do not accumulate internalized transferrin and so are different from the endosomes at the base of the epithelial cell and in the cell body of the neuron (Fuller and Simons, 1986;Hughson and Hopkins, 1990; Parton et al., 1992; Barroso and Sztul, 1994). Because the latter endosomes primarily recycle proteins involved in cell maintenance functions, we refer to them as housekeeping endosomes. Specialized endo- somes, on the other hand, operate in specialized regions of cells, such as the sub-apical cytoplasm, and appear to give rise to small tubulovesicular structures that fuse with the cell surface. Examples of such apicaUy recycling vesicles include those that contain the vasopressin-sensitive water channels in kidney collecting ductules (for review see Verkman, 1992), the gastrin-responsive H÷K+-ATPase in gastric parie...