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Aggresomes – a novel way to die slowly
headlines acid (PUFA)-enriched media inhibits the increase in concentration of cytoplasmic Ca21 and protein tyrosine phosphorylation that follows stimulation of GPI-anchored receptors such as CD59. CD59 remains clustered and associates with DRMs in PUFA-treated cells; by contrast, the Src-family protein tyrosine kinase Lck localizes diffusely and no longer sediments with
DRMs. How PUFA enrichment displaces Lck from the inner leaflet of rafts remains uncertain (through enhanced unsaturation of DRM lipids or changes in protein acylation?). Nonetheless, these data argue strongly that lipid-mediated association of CD59 and Lck is crucial to signal transduction in T cells. The results also provide possible insights into
clinical effects of PUFAs, such as T-cell suppression. 1
Stulnig, T. M., Berger, M., Sigmund, T., Raederstorff, D., Stockinger, H. and Waldhäusl, W. (1998) Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains, J. Cell Biol. 143, 637–644
The Golgi bypassed Plants have two, perhaps three, types of vacuoles with different functions, sometimes within a single cell. Each type is characterized by a different aquaporin, alpha, gamma or delta TIP (tonoplast intrinsic protein). These recently discovered extra destinations make targeting within the secretory system in plants much more complex than previously thought. This paper investigates the targeting determinants of two integral membrane proteins to find how proteins are delivered to their specific destination. The results suggest that plants have a route from the endoplasmic reticulum to the vacuole that is independent of the Golgi apparatus and suggest that transmembrane domains (TMDs) and cytoplasmic C-terminal tails are involved in this targeting. The lysosome-like lytic vacuole (LV) is characterized by gamma-TIP, low pH and soluble proteases such as the aleurain. A putative sorting receptor, BP-80, is believed to mediate traffic of soluble proteins from the Golgi towards this vacuole. BP-80 consists of a large, lumenal N-terminal domain, a single TMD and a short
C-terminal domain (CTD) with a motif that can bind to adaptins, consistent with the idea that clathrin is involved in its trafficking. Alpha-TIP on the other hand is associated with protein storage vacuoles (PSVs). It has six membrane spans and a short cytosolic C-terminus that is rich in proline and histidine. Previous work has shown that trafficking of this aquaporin is less sensitive to brefeldin A (BFA), an inhibitor of Golgi apparatus function. The soluble zymogen aleurain undergoes complex glycosylation in transit through the Golgi and is cleaved on arrival in the LV. Using a modified aleurain without vacuolar targeting determinants as a reporter protein, possible targeting signals in BP-80 and alpha TIP were investigated1. The resulting localization was determined biochemically, and constructs with modified C-termini were localized by immunofluoresence. In transgenic tobacco, a reporter protein with the TMD and CTD from BP-80 received complex glycan modification, underwent vacuolar processing and colocalized with tobacco BP-80 homologues at or near the LV.
The glycan modification and processing were BFA sensitive, so the reporter proteins travelled from the ER to the LV via the Golgi apparatus. By contrast, swapping the BP-80 TMD or CTD for the respective domain of alpha TIP prevented the normal glycan and proteolytic processing. Furthermore, colocalization of the reporter with BP-80 homologues was abolished, and it now colocalized with alpha TIP in a non-ER compartment, presumably the PSV. The results suggest that the alpha TIP CTD has active targeting information for the PSV, and that the route to the PSV branches from that to the LV in the ER. Since trafficking to the yeast vacuole and mammalian lysosome-like compartments has recently been shown to involve multiple routes, it will be interesting to see whether mammalian and yeast cells have pathways analogous to these emerging pathways in plants. 1
Jiang, L. and Rogers, J. C. (1998) Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways, J. Cell Biol. 143, 1183–1199
Aggresomes – a novel way to die slowly Proteins that enter the central vacuolar system at the rough endoplasmic reticulum (ER) and fail to assume their correct conformation are removed selectively by proteolysis, a process termed ER-associated degradation. In recent years, it has become clear that, in most cases, this pathway is mediated by the cytosolic proteasome, after retro-translocation of the substrate protein through the Sec61
130
translocation channel, deglycosylation in the case of glycoproteins, and, in many instances, ubiquitination. Now, Johnston et al. show that the cystic fibrosis transmembrane conductance regulator (CFTR) and presenilin-1, two multispanning membrane proteins that fold inefficiently in the ER, might accumulate after extrusion to the cytosol, where they build up stable, high-molecular-
weight, detergent-insoluble, multiubiquitinated aggregates, which they termed ‘aggresomes’1. The formation of these structures occurs either by overexpression of the wild-type or mutated substrate proteins (so that the proteasome activity might be saturated) or by inhibition of proteasomes. Using GFP-tagged CFTR as well as immunofluorescence analysis, the authors demonstrated that aggresome
trends in CELL BIOLOGY (Vol. 9) April 1999
headlines localization close to the microtubuleorganizing centre, but not the biogenesis of aggresomes itself, is dependent on intact microtubules. Furthermore, the intermediate filament protein vimentin redistributes upon formation of aggresomes, forming a cage-like structure around the aggregated proteins. It is proposed that the appearance of aggresomes is a general cellular
response to overexpression of proteins, when intracellular folding and degradation machineries are exhausted. Even though the proteins trapped in aggresomes seem to be rather stable, it will be interesting to learn about the dynamic nature of this novel structure, and about other components participating in its formation and/or present in the aggregates. Clearly, the importance of the
new findings is underscored by the role that protein aggregation is known to play in the pathogenesis of several diseases, including those characterized by neurodegeneration. 1
Johnston, J. A., Ward, C. L. and Kopito, R. R. (1998) Aggresomes: a cellular response to misfolded proteins, J. Cell Biol. 143, 1883–1898
Getting in is easy, but how to get out? In yeast, the heat-shock proteins Kar2p (BiP) and Ssa1p play essential roles in the import of nascent proteins into the ER by working together as ratchets or motors in the endoplasmic reticulum (ER) lumen and the cytosol, respectively. Defective imported proteins are scouted out by other ER chaperones such as calnexin (Cne1p) and prepared for export back into the cytosol, where they are degraded in proteasomes. Both the import of nascent proteins into the ER as well as the export of misfolded proteins from the ER require Kar2p and the Sec61p translocation complex, suggesting a mechanistic link between these two processes. To investigate this hypothesis, Brodsky et al.1 generated mutant alleles of some of the major players involved,
namely Cne1p, Kar2p and Ssa1p. By following the degradation of pro-a factor in vitro and A1PiZ in vivo, they could show that mutations in Kar2p inhibit this degradation and that Kar2p is required for export and for the recognition of degradation substrates. By contrast, mutations in Ssa1p had no effect on export or degradation, although they prevented protein import into the ER both in vivo and in vitro, showing that the essential nature of the role for Ssa1p for protein import does not extend to protein export. Finally, they observed a synthetic interaction between kar2 and cne1 at elevated temperatures. The authors suggest that misfolded proteins are captured by Cne1p in the ER lumen but must be handed over to
Kar2p for proper re-translocation. Mutations in either chaperone might compromise the release of the substrate and therefore abolish this handover. Taken together, the mechanisms for both the import of nascent polypeptides and for the export of misfolded proteins are distinct, although at least one player (Kar2p) is involved in both processes. 1
Brodsky, J., Werner, E. D., Dubas, M. E., Goeckeler, J. L., Kruse, K. B. and McCracken, A. A. (1999) The requirement for molecular chaperones during endoplasmic reticulumassociated protein degradation demonstrates that protein export and import are mechanistically distinct, J. Biol. Chem. 274, 3453–3460
Serum theorem The expression of genes in response to stimulation by serum or growth signals has been studied for a long time. Investigators usually focused on the genes that controlled cell proliferation because of the belief that cells respond transcriptionally to growth signals before dividing, although this approach was limited by the ability to analyse only a few genes at a time. With the introduction of DNA chips (i.e. DNA microarrays) that immobilize thousands of miscroscopic dots of genes (DNAs) on a glass slide, it is theoretically possible to analyse the entire set of genes in a cell simultaneously. Iyer et al.1 have used a cDNA microarray to look at the responses of 8613 different genes from human neonatal foreskin fibroblasts in culture to 10% fetal bovine serum stimulation after
48 hours of serum starvation. At different times after serum stimulation, cDNAs were made from the mRNA of the cells, labelled with the red fluorescent dye Cy5-dUTP and mixed with the green fluorescent dye Cy3-dUTPconjugated cDNA made from the mRNA of the cells from the quiescent state. After hybridization in the DNA microarrays, induced genes appear as red spots, repressed genes appear as green spots, while yellow spots represent genes whose expression remains fairly constant. Different classes of genes that have similar expression profiles were grouped together and microarray quantitations verified by a TaqMan 59-nuclease fluorigenic quantitative PCR assay. Among the genes that were induced, a large number were related
trends in CELL BIOLOGY (Vol. 9) April 1999
to the physiology of wound repair, such as clotting and activation of neutrophils. In addition, over 200 genes of unknown function had a regulated pattern of expression. Thus, fibroblasts appear to play a multifunctional role in response to serum. Where next? DNA chips provide a genome-scale analysis of gene expression. We can now view cellular responses as a whole, and eventually one might look at gene expression in a whole organism and see how different genes are coordinated.
1
Iyer, V. R. et al. (1999) The transcriptional program in the response of human fibroblasts to serum, Science 283, 83–87
131
DRMs. How PUFA enrichment displaces Lck from the inner leaflet of rafts remains uncertain (through enhanced unsaturation of DRM lipids or changes in protein acylation?). Nonetheless, these data argue strongly that lipid-mediated association of CD59 and Lck is crucial to signal transduction in T cells. The results also provide possible insights into
clinical effects of PUFAs, such as T-cell suppression. 1
Stulnig, T. M., Berger, M., Sigmund, T., Raederstorff, D., Stockinger, H. and Waldhäusl, W. (1998) Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains, J. Cell Biol. 143, 637–644
The Golgi bypassed Plants have two, perhaps three, types of vacuoles with different functions, sometimes within a single cell. Each type is characterized by a different aquaporin, alpha, gamma or delta TIP (tonoplast intrinsic protein). These recently discovered extra destinations make targeting within the secretory system in plants much more complex than previously thought. This paper investigates the targeting determinants of two integral membrane proteins to find how proteins are delivered to their specific destination. The results suggest that plants have a route from the endoplasmic reticulum to the vacuole that is independent of the Golgi apparatus and suggest that transmembrane domains (TMDs) and cytoplasmic C-terminal tails are involved in this targeting. The lysosome-like lytic vacuole (LV) is characterized by gamma-TIP, low pH and soluble proteases such as the aleurain. A putative sorting receptor, BP-80, is believed to mediate traffic of soluble proteins from the Golgi towards this vacuole. BP-80 consists of a large, lumenal N-terminal domain, a single TMD and a short
C-terminal domain (CTD) with a motif that can bind to adaptins, consistent with the idea that clathrin is involved in its trafficking. Alpha-TIP on the other hand is associated with protein storage vacuoles (PSVs). It has six membrane spans and a short cytosolic C-terminus that is rich in proline and histidine. Previous work has shown that trafficking of this aquaporin is less sensitive to brefeldin A (BFA), an inhibitor of Golgi apparatus function. The soluble zymogen aleurain undergoes complex glycosylation in transit through the Golgi and is cleaved on arrival in the LV. Using a modified aleurain without vacuolar targeting determinants as a reporter protein, possible targeting signals in BP-80 and alpha TIP were investigated1. The resulting localization was determined biochemically, and constructs with modified C-termini were localized by immunofluoresence. In transgenic tobacco, a reporter protein with the TMD and CTD from BP-80 received complex glycan modification, underwent vacuolar processing and colocalized with tobacco BP-80 homologues at or near the LV.
The glycan modification and processing were BFA sensitive, so the reporter proteins travelled from the ER to the LV via the Golgi apparatus. By contrast, swapping the BP-80 TMD or CTD for the respective domain of alpha TIP prevented the normal glycan and proteolytic processing. Furthermore, colocalization of the reporter with BP-80 homologues was abolished, and it now colocalized with alpha TIP in a non-ER compartment, presumably the PSV. The results suggest that the alpha TIP CTD has active targeting information for the PSV, and that the route to the PSV branches from that to the LV in the ER. Since trafficking to the yeast vacuole and mammalian lysosome-like compartments has recently been shown to involve multiple routes, it will be interesting to see whether mammalian and yeast cells have pathways analogous to these emerging pathways in plants. 1
Jiang, L. and Rogers, J. C. (1998) Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways, J. Cell Biol. 143, 1183–1199
Aggresomes – a novel way to die slowly Proteins that enter the central vacuolar system at the rough endoplasmic reticulum (ER) and fail to assume their correct conformation are removed selectively by proteolysis, a process termed ER-associated degradation. In recent years, it has become clear that, in most cases, this pathway is mediated by the cytosolic proteasome, after retro-translocation of the substrate protein through the Sec61
130
translocation channel, deglycosylation in the case of glycoproteins, and, in many instances, ubiquitination. Now, Johnston et al. show that the cystic fibrosis transmembrane conductance regulator (CFTR) and presenilin-1, two multispanning membrane proteins that fold inefficiently in the ER, might accumulate after extrusion to the cytosol, where they build up stable, high-molecular-
weight, detergent-insoluble, multiubiquitinated aggregates, which they termed ‘aggresomes’1. The formation of these structures occurs either by overexpression of the wild-type or mutated substrate proteins (so that the proteasome activity might be saturated) or by inhibition of proteasomes. Using GFP-tagged CFTR as well as immunofluorescence analysis, the authors demonstrated that aggresome
trends in CELL BIOLOGY (Vol. 9) April 1999
headlines localization close to the microtubuleorganizing centre, but not the biogenesis of aggresomes itself, is dependent on intact microtubules. Furthermore, the intermediate filament protein vimentin redistributes upon formation of aggresomes, forming a cage-like structure around the aggregated proteins. It is proposed that the appearance of aggresomes is a general cellular
response to overexpression of proteins, when intracellular folding and degradation machineries are exhausted. Even though the proteins trapped in aggresomes seem to be rather stable, it will be interesting to learn about the dynamic nature of this novel structure, and about other components participating in its formation and/or present in the aggregates. Clearly, the importance of the
new findings is underscored by the role that protein aggregation is known to play in the pathogenesis of several diseases, including those characterized by neurodegeneration. 1
Johnston, J. A., Ward, C. L. and Kopito, R. R. (1998) Aggresomes: a cellular response to misfolded proteins, J. Cell Biol. 143, 1883–1898
Getting in is easy, but how to get out? In yeast, the heat-shock proteins Kar2p (BiP) and Ssa1p play essential roles in the import of nascent proteins into the ER by working together as ratchets or motors in the endoplasmic reticulum (ER) lumen and the cytosol, respectively. Defective imported proteins are scouted out by other ER chaperones such as calnexin (Cne1p) and prepared for export back into the cytosol, where they are degraded in proteasomes. Both the import of nascent proteins into the ER as well as the export of misfolded proteins from the ER require Kar2p and the Sec61p translocation complex, suggesting a mechanistic link between these two processes. To investigate this hypothesis, Brodsky et al.1 generated mutant alleles of some of the major players involved,
namely Cne1p, Kar2p and Ssa1p. By following the degradation of pro-a factor in vitro and A1PiZ in vivo, they could show that mutations in Kar2p inhibit this degradation and that Kar2p is required for export and for the recognition of degradation substrates. By contrast, mutations in Ssa1p had no effect on export or degradation, although they prevented protein import into the ER both in vivo and in vitro, showing that the essential nature of the role for Ssa1p for protein import does not extend to protein export. Finally, they observed a synthetic interaction between kar2 and cne1 at elevated temperatures. The authors suggest that misfolded proteins are captured by Cne1p in the ER lumen but must be handed over to
Kar2p for proper re-translocation. Mutations in either chaperone might compromise the release of the substrate and therefore abolish this handover. Taken together, the mechanisms for both the import of nascent polypeptides and for the export of misfolded proteins are distinct, although at least one player (Kar2p) is involved in both processes. 1
Brodsky, J., Werner, E. D., Dubas, M. E., Goeckeler, J. L., Kruse, K. B. and McCracken, A. A. (1999) The requirement for molecular chaperones during endoplasmic reticulumassociated protein degradation demonstrates that protein export and import are mechanistically distinct, J. Biol. Chem. 274, 3453–3460
Serum theorem The expression of genes in response to stimulation by serum or growth signals has been studied for a long time. Investigators usually focused on the genes that controlled cell proliferation because of the belief that cells respond transcriptionally to growth signals before dividing, although this approach was limited by the ability to analyse only a few genes at a time. With the introduction of DNA chips (i.e. DNA microarrays) that immobilize thousands of miscroscopic dots of genes (DNAs) on a glass slide, it is theoretically possible to analyse the entire set of genes in a cell simultaneously. Iyer et al.1 have used a cDNA microarray to look at the responses of 8613 different genes from human neonatal foreskin fibroblasts in culture to 10% fetal bovine serum stimulation after
48 hours of serum starvation. At different times after serum stimulation, cDNAs were made from the mRNA of the cells, labelled with the red fluorescent dye Cy5-dUTP and mixed with the green fluorescent dye Cy3-dUTPconjugated cDNA made from the mRNA of the cells from the quiescent state. After hybridization in the DNA microarrays, induced genes appear as red spots, repressed genes appear as green spots, while yellow spots represent genes whose expression remains fairly constant. Different classes of genes that have similar expression profiles were grouped together and microarray quantitations verified by a TaqMan 59-nuclease fluorigenic quantitative PCR assay. Among the genes that were induced, a large number were related
trends in CELL BIOLOGY (Vol. 9) April 1999
to the physiology of wound repair, such as clotting and activation of neutrophils. In addition, over 200 genes of unknown function had a regulated pattern of expression. Thus, fibroblasts appear to play a multifunctional role in response to serum. Where next? DNA chips provide a genome-scale analysis of gene expression. We can now view cellular responses as a whole, and eventually one might look at gene expression in a whole organism and see how different genes are coordinated.
1
Iyer, V. R. et al. (1999) The transcriptional program in the response of human fibroblasts to serum, Science 283, 83–87
131