Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmen... more Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion, impaired cell migration and failure to undergo Epidermal to Mesenchymal Transition (EMT). However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock out Mouse Embryonic Fibroblasts (MEFs) to investigate its endocytic function. We found that kazrin depletion delays perinuclear enrichment of internalized material, indicating a role in endocytic traffic from Early (EE) to Recycling Endosomes (REs). Consistently, we found that the C-terminal domain of kazrin C, predicted to be an Intrinsically Disordered Region (IDR), directly interacts with several components of the EEs, and that kazrin depletion impair...
Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport ... more Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.
Kazrin C regulates endocytic recycling pathways and controls cell migration
Trabajo presentado en el EMBO Workshop: Cell polarity and membrane dynamics, celebrado en Sant Fe... more Trabajo presentado en el EMBO Workshop: Cell polarity and membrane dynamics, celebrado en Sant Feliu de Guixols, Girona, del 4 al 9 de junio de 2017Kazrin C was identified in a screening for human proteins that affect endocytic traffic when overexpressed. Indeed, its depletion delays the transfer of Transferrin from sorting to the Rab11 containing endosomes, whereas it accelerates a short loop recycling pathway. Kazrin C is localized at endosomes containing Transferrin and Rab4 and it directly interacts with components of the clathrin coat, such as clathrin and ¿-adaptin, and with components of the actin cytoskeleton, such as the ARP2/3 complex and WASH. In addition, it interacts with several phosphoinositides, which have a role in the regulation of endocytic trafficking. Our hypothesis is that Kazrin C favours the long recycling route in opposition to the short recycling route, either by controlling actin polymerization, by influencing the endosomal phosphoinositide content and/or by promoting loading of cargo into AP-1 and clathrin transport intermediates. The balance between these two routes is known to be important in the control of cell migration: ¿5ß1 integrin is recycled through the long route and its presence at the plasma membrane promotes random migration, while ¿vß3 follows the short route and promotes directionally persistent migration. In agreement with this and our hypothesis, we have observed an increased persistance in the migration of Kazrin C depleted cells when compared to control cells.N
OSBP-mediated ER contact with endocytic sites initiates actin polymerization
Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodell... more Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodelling and organization”, celebrado en Roscoff (Francia), del 3 al 7 de abril de 2017Oxysterol Binding Proteins (OSBP) are a family of conserved lipid binding proteins, enriched at endoplasmic reticulum (ER) contacts sites. OSBPs promote non-vesicular lipid transport to other organelles and work as lipid sensors in the context of multiple cellular tasks, but the determinants of their distinct localization and function are still not understood. Using a combination of Time Resolved Electron Microscopy (TREM) and life-cell imaging in yeast, we demonstrate that the endocytic invaginations associate with the cortical endoplasmic reticulum as they mature, and that this association requires the OSBPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Using mutations that specifically disrupt the myosin-I/OSBP/VAP link, as well as...
1 Current Biology, Volume 23 Supplemental Information Cell-to-Cell Heterogeneity in Lipid Droplets Suggests a Mechanism to Reduce Lipotoxicity
Figure S1. Related to Figure 1 (A) Triglycerides measured by enzymatic methods or by flow cytomet... more Figure S1. Related to Figure 1 (A) Triglycerides measured by enzymatic methods or by flow cytometry with Nile red and BODIPY 493/503 in Non-loaded, Loaded and Loaded cells treated with TriacsinC 10µM (Loaded+TRC). Values are referred to Non-loaded cells. (B) Heterogeneity in LD content in Non-loaded and cells Loaded with 175µg/ml FA from different cell lines. Red bars correspond to hepatic cell lines. Data represent mean ± SEM of 3 independent experiments.
Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily con... more Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.
As cells migrate and experience forces from their surroundings, they constantly undergo mechanica... more As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physi...
Deciphering Protein Dynamics during Endocytic Budding by Time Resolved Electron Microscopy
Trabajo presentado en el 28th Annual Symposium of The Protein Society, celebrado en San Diego, Ca... more Trabajo presentado en el 28th Annual Symposium of The Protein Society, celebrado en San Diego, California, Estados Unidos, del 27 al 30 de julio de 2014
F1000 - Post-publication peer review of the biomedical literature, 2011
The large GTPase dynamin assembles into higher order structures that are thought to promote endoc... more The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.
During maize seed development, endosperm cells synthesize large amounts of storage proteins,- ,-,... more During maize seed development, endosperm cells synthesize large amounts of storage proteins,- ,-, andzeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize-zein gene through the introduction of lysine-rich (Pro-Lys) n coding sequences at different sites of the-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich-zein constructs under the control of the 1.7 kb-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys) n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the-zein, high levels of protein were observed. In contrast, when (Pro-Lys) n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified-zeins accumulate within these organelles and co-localized with endogenous-and-zeins. The studies reported here show the feasibility of manipulating the-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.
Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1... more Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipidbinding domain. Some myosins-I have an additional C-terminal extension (C ext) that promotes Arp2/3 complexdependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulinrelated light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C ext. The TH1 domain prevented binding of the Myo5 C ext to the yeast WIP homologue Vrp1, Myo5 C ext-induced actin polymerization and recruitment of the Myo5 C ext to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C ext. Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.
In order to examine the role of cysteine (Cys)rich domains in the accumulation of maize (Zea mays... more In order to examine the role of cysteine (Cys)rich domains in the accumulation of maize (Zea mays L.) 7-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of 7-zein and of two truncated forms of 7-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the 7-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire 7-zein and both truncated forms of the 7-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for 7-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for 7-zein targeting on the endoplasmic reticulum.
Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of... more Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein i. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.
Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and d... more Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed β-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of α-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to inte...
Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. I... more Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-I Myo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites.
Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enric... more Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.
Localized synthesis and transport of sterol at ER endocytic contact sites initiates actin polymerization
Trabajo presentado en las Lysosomes and Endocytosis Gordon Research Conference: Molecules, Pathwa... more Trabajo presentado en las Lysosomes and Endocytosis Gordon Research Conference: Molecules, Pathways, and Physiology of Endosomes, Lysosomes and Lysosome-Related Organelles, celebradas en Andover, New Hampshire (USA), del 17 al 22 de junio de 2018
Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Th... more Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p’s function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-�CT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and func...
Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmen... more Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion, impaired cell migration and failure to undergo Epidermal to Mesenchymal Transition (EMT). However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock out Mouse Embryonic Fibroblasts (MEFs) to investigate its endocytic function. We found that kazrin depletion delays perinuclear enrichment of internalized material, indicating a role in endocytic traffic from Early (EE) to Recycling Endosomes (REs). Consistently, we found that the C-terminal domain of kazrin C, predicted to be an Intrinsically Disordered Region (IDR), directly interacts with several components of the EEs, and that kazrin depletion impair...
Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport ... more Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.
Kazrin C regulates endocytic recycling pathways and controls cell migration
Trabajo presentado en el EMBO Workshop: Cell polarity and membrane dynamics, celebrado en Sant Fe... more Trabajo presentado en el EMBO Workshop: Cell polarity and membrane dynamics, celebrado en Sant Feliu de Guixols, Girona, del 4 al 9 de junio de 2017Kazrin C was identified in a screening for human proteins that affect endocytic traffic when overexpressed. Indeed, its depletion delays the transfer of Transferrin from sorting to the Rab11 containing endosomes, whereas it accelerates a short loop recycling pathway. Kazrin C is localized at endosomes containing Transferrin and Rab4 and it directly interacts with components of the clathrin coat, such as clathrin and ¿-adaptin, and with components of the actin cytoskeleton, such as the ARP2/3 complex and WASH. In addition, it interacts with several phosphoinositides, which have a role in the regulation of endocytic trafficking. Our hypothesis is that Kazrin C favours the long recycling route in opposition to the short recycling route, either by controlling actin polymerization, by influencing the endosomal phosphoinositide content and/or by promoting loading of cargo into AP-1 and clathrin transport intermediates. The balance between these two routes is known to be important in the control of cell migration: ¿5ß1 integrin is recycled through the long route and its presence at the plasma membrane promotes random migration, while ¿vß3 follows the short route and promotes directionally persistent migration. In agreement with this and our hypothesis, we have observed an increased persistance in the migration of Kazrin C depleted cells when compared to control cells.N
OSBP-mediated ER contact with endocytic sites initiates actin polymerization
Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodell... more Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodelling and organization”, celebrado en Roscoff (Francia), del 3 al 7 de abril de 2017Oxysterol Binding Proteins (OSBP) are a family of conserved lipid binding proteins, enriched at endoplasmic reticulum (ER) contacts sites. OSBPs promote non-vesicular lipid transport to other organelles and work as lipid sensors in the context of multiple cellular tasks, but the determinants of their distinct localization and function are still not understood. Using a combination of Time Resolved Electron Microscopy (TREM) and life-cell imaging in yeast, we demonstrate that the endocytic invaginations associate with the cortical endoplasmic reticulum as they mature, and that this association requires the OSBPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Using mutations that specifically disrupt the myosin-I/OSBP/VAP link, as well as...
1 Current Biology, Volume 23 Supplemental Information Cell-to-Cell Heterogeneity in Lipid Droplets Suggests a Mechanism to Reduce Lipotoxicity
Figure S1. Related to Figure 1 (A) Triglycerides measured by enzymatic methods or by flow cytomet... more Figure S1. Related to Figure 1 (A) Triglycerides measured by enzymatic methods or by flow cytometry with Nile red and BODIPY 493/503 in Non-loaded, Loaded and Loaded cells treated with TriacsinC 10µM (Loaded+TRC). Values are referred to Non-loaded cells. (B) Heterogeneity in LD content in Non-loaded and cells Loaded with 175µg/ml FA from different cell lines. Red bars correspond to hepatic cell lines. Data represent mean ± SEM of 3 independent experiments.
Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily con... more Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.
As cells migrate and experience forces from their surroundings, they constantly undergo mechanica... more As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physi...
Deciphering Protein Dynamics during Endocytic Budding by Time Resolved Electron Microscopy
Trabajo presentado en el 28th Annual Symposium of The Protein Society, celebrado en San Diego, Ca... more Trabajo presentado en el 28th Annual Symposium of The Protein Society, celebrado en San Diego, California, Estados Unidos, del 27 al 30 de julio de 2014
F1000 - Post-publication peer review of the biomedical literature, 2011
The large GTPase dynamin assembles into higher order structures that are thought to promote endoc... more The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.
During maize seed development, endosperm cells synthesize large amounts of storage proteins,- ,-,... more During maize seed development, endosperm cells synthesize large amounts of storage proteins,- ,-, andzeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize-zein gene through the introduction of lysine-rich (Pro-Lys) n coding sequences at different sites of the-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich-zein constructs under the control of the 1.7 kb-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys) n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the-zein, high levels of protein were observed. In contrast, when (Pro-Lys) n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified-zeins accumulate within these organelles and co-localized with endogenous-and-zeins. The studies reported here show the feasibility of manipulating the-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.
Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1... more Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipidbinding domain. Some myosins-I have an additional C-terminal extension (C ext) that promotes Arp2/3 complexdependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulinrelated light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C ext. The TH1 domain prevented binding of the Myo5 C ext to the yeast WIP homologue Vrp1, Myo5 C ext-induced actin polymerization and recruitment of the Myo5 C ext to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C ext. Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.
In order to examine the role of cysteine (Cys)rich domains in the accumulation of maize (Zea mays... more In order to examine the role of cysteine (Cys)rich domains in the accumulation of maize (Zea mays L.) 7-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of 7-zein and of two truncated forms of 7-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the 7-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire 7-zein and both truncated forms of the 7-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for 7-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for 7-zein targeting on the endoplasmic reticulum.
Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of... more Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein i. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.
Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and d... more Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed β-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of α-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to inte...
Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. I... more Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-I Myo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites.
Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enric... more Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.
Localized synthesis and transport of sterol at ER endocytic contact sites initiates actin polymerization
Trabajo presentado en las Lysosomes and Endocytosis Gordon Research Conference: Molecules, Pathwa... more Trabajo presentado en las Lysosomes and Endocytosis Gordon Research Conference: Molecules, Pathways, and Physiology of Endosomes, Lysosomes and Lysosome-Related Organelles, celebradas en Andover, New Hampshire (USA), del 17 al 22 de junio de 2018
Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Th... more Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p’s function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-�CT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and func...
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