- Email: [email protected]
Study of shiga toxin B-fragment transport from early endosomes to the golgi apparatus
120
Biology of the Cell (1998) 90, 109-l 30
LEBORGNE-CASTEL Nathalie and DENECKE Jiirgen The Plant + Present Bwrgogrte,
*, VAN DOOREN
Edith
Laboratorv. Universitvof York.Heslinnton. York Y01 5DD. UK. addresi’: Laboraioire de k&%&e Celftibire,~&vers!te de 9 avenueAlain Savary, B.P. %4 (21 Ul I Dijo~ cedex, France
The Iuminal binding protein BiP, a resident of the endoplasmic reticulum (ER), is a member of a wide class of protein termed molecular chaperone. It is structurally and functionally related to cytosolii HSP70 but differing in the presence of a signal peptide and an ER retention signal (K/HDEL). In tobacco, BiP is encoded by a multigene family and at least out member is able to complement a yeast mutant indicating to a same functional role (Denecke J., Grjldman M. H., Demolder .I., Seurick J. and Botterman J. (1991), Plonf Ceil 3, 1025-1035). Although BiP is present in detectable amount under normal conditions, it can be induced by a variety of stress(e.g. tunicamycin) rest&g in mtiokled secretoryproteins (Denecke J. and Vi&e A. (1995). Methods in C&l Biol. 24, 335-348). Biochemical evidence shows its implication in the process of protein folding due to its ability to bind to polypeptides in the ER lumen (Pedrazzini E. and Vitale A. (1996), Plant Physid. Biochem. 34, 207-216) but the role of BiP in tbc quality contra1 by the ER retention of malfolded proreins is not clearly established. We were interested to alter expression of this molecular chaperone to provide more information about its function in secretion and tmhsport of proteins in plants. We set up a.functional assay for BiP based on the comparison of transient protein synthesis in the cytosol (GUS) and on the rough ER (barley n-amylase) after electroporation of tobiT protoplasrs in presence of corresponding genes. Co-nansfection experiments were carried out using both 1) a plasmid containing GUS and a-amylase genei and 2) a plasmid containing BiP construct. A positive effect of transient overexpression of BiP on the synthesis of a-amylase under ER stresshas been observed. Then, we have generated transgenic tobacco plants exhibiting different levels of BiP or expressing BiP mutants which will be used as &ls to better understand Sip regul&on, protein synthesis and proteins transport in plants. This work was supported by H. C. M. grant (ERBCHRX-CIY4-0590)
THE LD DlJlttW
MALlARO Fr&d&ic, TENZA Dan&, SALAMERO Jean, ANTONY Claude, GOUD Bruno and JOHANNES Ludger CNRS UMR 144, lfistitut Curie, Paris, France
We have previously shown that in HeLa cells, Shiga toxin B-fragment 13 transported retrogladely from the plasma membrane via eniiosomes and the Golgi apparatus IO the endoplasmic reticulum (Johannes et al. 11997}, J Biol. Chem., 272,19554- 19561). Here we found that at 19.5’C. B--fragment transport to &r Golg: apparatus was inhibited. We then compared by confocal andimmunoelectron microscopy ori both fixed and living celts the transport ol’ B-fragment from the 19.S’C compartment to the Golgi appartrtus to transport of several marker proteins of the endocytic pathway. At l%YC, the B-fragment colocalized compfetely with transferrin in early/recycling endosomes. In con&& the colocalization with -fluid phase markers-(3 kD dextran and BSA-gold) was less complete, and internalized EGF, which inassociation with its receptor is transported to lyysosomes,was always formd adjacent to B-fragment positive compartm+ with no overlap between both markers. These data suggest that at 19.5’C, B-fragment is sorted tu early/recycling endosomes,away from marker proteins that are destined fm late endosomesflvsosomes.Uoon shift to 37°C. the B-fraamem is raaidlv transported to th;Goigi apparirus (lo-20 min). Doting this”gransport,io a’ fragment specific. label is found in BSA-gold containing poly-vesicular (late) endosomes, suggesting that B-fragment transporf tu the Golgiapparatusoccurs from &&ly/reEycling endos&mes.This hypothesis is fun& strengthened by the finding that in morphological a?d biochemicalexueriments, several drurs (bafilomvcin Al.~-concanamvcin R. anti cy&chalasin’D) with escabsshkd effect; on v&cular transp&t in the lan endocytic pathway did nor affect B-fragment transport to the Cotgi apparatus. In contrast, in the presence of brefeldin A, the B-fragment accumulated in tubular structures that also contained a marker protemuf early endosomes, the rransferrin receptor, while EGF was efficiently degraded in tysosomes, indicating that the transport route to late endosomesflysosomeswas open. Taken together, these resuftsare consislem wirh the bypothesk 6: Shiga toxin B-fragment is EanspOI’ted directly from early/recycling endosomes to the Gotgi apparatus. This hovel transport route could alsrt be used by cellular proteins, as deduced from our finding that TCiN3X,+a protein which ar steadystate is concentrated in the TGN but which has been reported to cycle through the plasma membrane, c&cakes with tkk Bfragment on its aansport to the Golgi apparatus.
DMDLOG,
da SILVA CONCEICAO A&and&‘, MARTY&WARS Dani&te$ ARRAlJlT Sar&a§, SANDERFOOT Anton A. *, LEVANOFW Uanna#, GAL/t/ Gad#, MARTY Fran&, and RAfKffELNatasha V. 5 Laboratoife de phyt@B&gfe Celktlaire, UPR ES 469, BP 400, Unnlersrtti de Bourgogne, 2101 I Djoo Cedex, Fraace; MWOOE Plant Res.-LabJvWigan State university, East Lansing, MI 488244-1312, USA; # Dept. Plant G_en&ics, The Weizrnarm hsfifute of Science, R&mot 76100, Israel
AWRAN Nathalie, MAROUX Suzanne
MASSEY-HARRDCHE
Dominique,
LBBN, Fat. St It&e,
case 342,13397
Marseille cedex 20, francs
l
l
Targeting of protein to the vacuole/lysosome is a multi-step process that appears to have conserved features between mammalian, yeast, and plant cells. Soluble vacuolarflysosomal proteins are believed to be bound by transmembrane cargo-receptors in the rrans Golgi network (TGN) that redirects these proteins into clarhrin-coated vesicles. These vesicles then appear to be transported to the prevacuolelendosome by a trafficking machinery similar to other vesicle targeting steps. Two likely members of this trafficking machinery have been characterized from Arabidopsis rhaKarza : AtPEP12p, a t-SNARE that resides on a post-Go@ compartment (da Silva Concei$ao A. a al. (1997), Plant Cell, 9, 571-582), and ALELP, a protein that shares common features with mammalian and yeast rransmembrane cargo receptors (Ahmed S.U. et al. (19973, Plant Physiol., 114,325-336). AtPEP12p is a syntaxin homoiog which has been cloned from 4rabidopsis rhaliana by functional complementation oaf the yeast pep1 2 nutant (Bassham D.C. et ai. (19%). Proc. Natl. Acac. Sci. USA, 92, 72627266). The Yeast PEP12o has a function in the transoori of vacuolar ?ydr&ases &d is thoug’ht to be associated with ai endosome-tike :ompartment (Becherer K.A. et al. (1996). Mol. Cell Biol., 7, 579-594). Consistently with a similar role for homologous proteins in plants, our -esults indicate rhat AtPEP12p is C-terminal anchored to the membranes of 1 post-Golgi compartment of Arabidqwis root cells. In this report, we pursue the characterization of the AtPEP12p :ompartmenr in Arabidopsis roofs and throughout seed development. In ‘oats, &PEPlZp is associated with membrane-bound reticuls-tubular iuuctures in the proximity to small vacuoles. Furthermore; subdellular ‘ractionations indicate that AtPEPlZp does not localize to ER, Golgi apparatus, tonOphst OF plasma membrane. In Contrast throughout seed ievelopment, rather than being associated with the surrounding membrane tf any organelle, AtPEP12p is fou&dispersed in the lumeti ofprotein lodies. These surprising results should be interpreted in the coi)text of .ecycIingmembrane components during the,formation of storage organelles
Ten years after the discovery of annexins (Anx) and in spite of t&e multiplicity of functions that these proteins seem -to perform in r&o, their in vivo roles are still unknown, However their intervention in the intracellultir membrane traffic is more and .more evident, for example Anx II is involved in hanscytosis of heijatocytes in culture and Anx XIIIb in the sorting of vesicles to the apical plasma membrane in MDCK cells. Few in situ localiiations of xnnexins are known however this point seems important to us in regard to their roles in viva. The isolation of several annexinS (I, II, IV, VI and XII(I) was carried out and several specific monoclonal antibodies were obtained. The tissular distribution, the cellular and the subcelhjhr localization of these different annexins by immlinofluerescewe~on thin frozen sections of organs was then studied. In the present work, the results obtained on intestine, tiver and pancreas are presented here. In enterocytes only annexins II, IV and XIII are expressed. These annexins are present at the basolateral domain of this cell type, this localization is strict for Anx IV when the presence of Anx II .is also revealed in the upperpart of the terminal web, and for Anx XIII at the Z/3 inferior of b&t border microvtlli. In rabbit, Anx XIII is absolutely specific for the small intestine. In colonocytes Anx I is localized to the basalateral membrane, Anx II and IV have the same localization than in enterocytes. In hepatcqtes Anx II, IV and VI are present Bt the sinusoi;;ial (basal) and I.&era1 domains. Anx II is also found in the apical pole. and Anx IV and vl on some apical vesicles concentrated around the bile canaliculi. In pancreatic acinar cells Anx IV and VI are present in the basolateral domain and Anx II is present on some zymogen granules. In conch&ion this study shows that theexpression oi’ annexins is finely regulated, and that they are mostly localized at the basolateral domain of polarized cells. This localization at the pIasma membrane
of cells is consistent
with an implication
last steps of protein exportation.
Meeting of the French Society of Cell Biology, 8-10 March 1998, lnstitut Curie, Pars, France
of annexins
in the
Biology of the Cell (1998) 90, 109-l 30
LEBORGNE-CASTEL Nathalie and DENECKE Jiirgen The Plant + Present Bwrgogrte,
*, VAN DOOREN
Edith
Laboratorv. Universitvof York.Heslinnton. York Y01 5DD. UK. addresi’: Laboraioire de k&%&e Celftibire,~&vers!te de 9 avenueAlain Savary, B.P. %4 (21 Ul I Dijo~ cedex, France
The Iuminal binding protein BiP, a resident of the endoplasmic reticulum (ER), is a member of a wide class of protein termed molecular chaperone. It is structurally and functionally related to cytosolii HSP70 but differing in the presence of a signal peptide and an ER retention signal (K/HDEL). In tobacco, BiP is encoded by a multigene family and at least out member is able to complement a yeast mutant indicating to a same functional role (Denecke J., Grjldman M. H., Demolder .I., Seurick J. and Botterman J. (1991), Plonf Ceil 3, 1025-1035). Although BiP is present in detectable amount under normal conditions, it can be induced by a variety of stress(e.g. tunicamycin) rest&g in mtiokled secretoryproteins (Denecke J. and Vi&e A. (1995). Methods in C&l Biol. 24, 335-348). Biochemical evidence shows its implication in the process of protein folding due to its ability to bind to polypeptides in the ER lumen (Pedrazzini E. and Vitale A. (1996), Plant Physid. Biochem. 34, 207-216) but the role of BiP in tbc quality contra1 by the ER retention of malfolded proreins is not clearly established. We were interested to alter expression of this molecular chaperone to provide more information about its function in secretion and tmhsport of proteins in plants. We set up a.functional assay for BiP based on the comparison of transient protein synthesis in the cytosol (GUS) and on the rough ER (barley n-amylase) after electroporation of tobiT protoplasrs in presence of corresponding genes. Co-nansfection experiments were carried out using both 1) a plasmid containing GUS and a-amylase genei and 2) a plasmid containing BiP construct. A positive effect of transient overexpression of BiP on the synthesis of a-amylase under ER stresshas been observed. Then, we have generated transgenic tobacco plants exhibiting different levels of BiP or expressing BiP mutants which will be used as &ls to better understand Sip regul&on, protein synthesis and proteins transport in plants. This work was supported by H. C. M. grant (ERBCHRX-CIY4-0590)
THE LD DlJlttW
MALlARO Fr&d&ic, TENZA Dan&, SALAMERO Jean, ANTONY Claude, GOUD Bruno and JOHANNES Ludger CNRS UMR 144, lfistitut Curie, Paris, France
We have previously shown that in HeLa cells, Shiga toxin B-fragment 13 transported retrogladely from the plasma membrane via eniiosomes and the Golgi apparatus IO the endoplasmic reticulum (Johannes et al. 11997}, J Biol. Chem., 272,19554- 19561). Here we found that at 19.5’C. B--fragment transport to &r Golg: apparatus was inhibited. We then compared by confocal andimmunoelectron microscopy ori both fixed and living celts the transport ol’ B-fragment from the 19.S’C compartment to the Golgi appartrtus to transport of several marker proteins of the endocytic pathway. At l%YC, the B-fragment colocalized compfetely with transferrin in early/recycling endosomes. In con&& the colocalization with -fluid phase markers-(3 kD dextran and BSA-gold) was less complete, and internalized EGF, which inassociation with its receptor is transported to lyysosomes,was always formd adjacent to B-fragment positive compartm+ with no overlap between both markers. These data suggest that at 19.5’C, B-fragment is sorted tu early/recycling endosomes,away from marker proteins that are destined fm late endosomesflvsosomes.Uoon shift to 37°C. the B-fraamem is raaidlv transported to th;Goigi apparirus (lo-20 min). Doting this”gransport,io a’ fragment specific. label is found in BSA-gold containing poly-vesicular (late) endosomes, suggesting that B-fragment transporf tu the Golgiapparatusoccurs from &&ly/reEycling endos&mes.This hypothesis is fun& strengthened by the finding that in morphological a?d biochemicalexueriments, several drurs (bafilomvcin Al.~-concanamvcin R. anti cy&chalasin’D) with escabsshkd effect; on v&cular transp&t in the lan endocytic pathway did nor affect B-fragment transport to the Cotgi apparatus. In contrast, in the presence of brefeldin A, the B-fragment accumulated in tubular structures that also contained a marker protemuf early endosomes, the rransferrin receptor, while EGF was efficiently degraded in tysosomes, indicating that the transport route to late endosomesflysosomeswas open. Taken together, these resuftsare consislem wirh the bypothesk 6: Shiga toxin B-fragment is EanspOI’ted directly from early/recycling endosomes to the Gotgi apparatus. This hovel transport route could alsrt be used by cellular proteins, as deduced from our finding that TCiN3X,+a protein which ar steadystate is concentrated in the TGN but which has been reported to cycle through the plasma membrane, c&cakes with tkk Bfragment on its aansport to the Golgi apparatus.
DMDLOG,
da SILVA CONCEICAO A&and&‘, MARTY&WARS Dani&te$ ARRAlJlT Sar&a§, SANDERFOOT Anton A. *, LEVANOFW Uanna#, GAL/t/ Gad#, MARTY Fran&, and RAfKffELNatasha V. 5 Laboratoife de phyt@B&gfe Celktlaire, UPR ES 469, BP 400, Unnlersrtti de Bourgogne, 2101 I Djoo Cedex, Fraace; MWOOE Plant Res.-LabJvWigan State university, East Lansing, MI 488244-1312, USA; # Dept. Plant G_en&ics, The Weizrnarm hsfifute of Science, R&mot 76100, Israel
AWRAN Nathalie, MAROUX Suzanne
MASSEY-HARRDCHE
Dominique,
LBBN, Fat. St It&e,
case 342,13397
Marseille cedex 20, francs
l
l
Targeting of protein to the vacuole/lysosome is a multi-step process that appears to have conserved features between mammalian, yeast, and plant cells. Soluble vacuolarflysosomal proteins are believed to be bound by transmembrane cargo-receptors in the rrans Golgi network (TGN) that redirects these proteins into clarhrin-coated vesicles. These vesicles then appear to be transported to the prevacuolelendosome by a trafficking machinery similar to other vesicle targeting steps. Two likely members of this trafficking machinery have been characterized from Arabidopsis rhaKarza : AtPEP12p, a t-SNARE that resides on a post-Go@ compartment (da Silva Concei$ao A. a al. (1997), Plant Cell, 9, 571-582), and ALELP, a protein that shares common features with mammalian and yeast rransmembrane cargo receptors (Ahmed S.U. et al. (19973, Plant Physiol., 114,325-336). AtPEP12p is a syntaxin homoiog which has been cloned from 4rabidopsis rhaliana by functional complementation oaf the yeast pep1 2 nutant (Bassham D.C. et ai. (19%). Proc. Natl. Acac. Sci. USA, 92, 72627266). The Yeast PEP12o has a function in the transoori of vacuolar ?ydr&ases &d is thoug’ht to be associated with ai endosome-tike :ompartment (Becherer K.A. et al. (1996). Mol. Cell Biol., 7, 579-594). Consistently with a similar role for homologous proteins in plants, our -esults indicate rhat AtPEP12p is C-terminal anchored to the membranes of 1 post-Golgi compartment of Arabidqwis root cells. In this report, we pursue the characterization of the AtPEP12p :ompartmenr in Arabidopsis roofs and throughout seed development. In ‘oats, &PEPlZp is associated with membrane-bound reticuls-tubular iuuctures in the proximity to small vacuoles. Furthermore; subdellular ‘ractionations indicate that AtPEPlZp does not localize to ER, Golgi apparatus, tonOphst OF plasma membrane. In Contrast throughout seed ievelopment, rather than being associated with the surrounding membrane tf any organelle, AtPEP12p is fou&dispersed in the lumeti ofprotein lodies. These surprising results should be interpreted in the coi)text of .ecycIingmembrane components during the,formation of storage organelles
Ten years after the discovery of annexins (Anx) and in spite of t&e multiplicity of functions that these proteins seem -to perform in r&o, their in vivo roles are still unknown, However their intervention in the intracellultir membrane traffic is more and .more evident, for example Anx II is involved in hanscytosis of heijatocytes in culture and Anx XIIIb in the sorting of vesicles to the apical plasma membrane in MDCK cells. Few in situ localiiations of xnnexins are known however this point seems important to us in regard to their roles in viva. The isolation of several annexinS (I, II, IV, VI and XII(I) was carried out and several specific monoclonal antibodies were obtained. The tissular distribution, the cellular and the subcelhjhr localization of these different annexins by immlinofluerescewe~on thin frozen sections of organs was then studied. In the present work, the results obtained on intestine, tiver and pancreas are presented here. In enterocytes only annexins II, IV and XIII are expressed. These annexins are present at the basolateral domain of this cell type, this localization is strict for Anx IV when the presence of Anx II .is also revealed in the upperpart of the terminal web, and for Anx XIII at the Z/3 inferior of b&t border microvtlli. In rabbit, Anx XIII is absolutely specific for the small intestine. In colonocytes Anx I is localized to the basalateral membrane, Anx II and IV have the same localization than in enterocytes. In hepatcqtes Anx II, IV and VI are present Bt the sinusoi;;ial (basal) and I.&era1 domains. Anx II is also found in the apical pole. and Anx IV and vl on some apical vesicles concentrated around the bile canaliculi. In pancreatic acinar cells Anx IV and VI are present in the basolateral domain and Anx II is present on some zymogen granules. In conch&ion this study shows that theexpression oi’ annexins is finely regulated, and that they are mostly localized at the basolateral domain of polarized cells. This localization at the pIasma membrane
of cells is consistent
with an implication
last steps of protein exportation.
Meeting of the French Society of Cell Biology, 8-10 March 1998, lnstitut Curie, Pars, France
of annexins
in the