Search

Coat protein I (COPI) and Coat protein II (COPII) coated vesicles mediate protein transport in the early secretory pathway. Although several components of COPII vesicles have been shown to have an essential role in Arabidopsis gametogenesis, the function of COPI components in gametogenesis has not been studied in detail. COPI consists of a heptameric complex made of α, β, β', γ, δ, ɛ, and ζ-COP subunits and most subunits have several isoforms in Arabidopsis. We have found that two isoforms of the β'-COP subunit, β'1-COP and β'2-COP, are required for female and male gametophyte development. Reciprocal crosses between wild type plants and plants heterozygous for T-DNA insertions in β'1-COP and β'2-COP showed that β'1β'2-cop gametophytes are not transmitted., (© 2023. The Author(s).)

Coat protein I (COPI) is necessary for intra-Golgi transport and retrograde transport from the Golgi apparatus back to the endoplasmic reticulum. The key component of the COPI coat is the coatomer complex, which is composed of seven subunits (α/β/β'/γ/δ/ε/ζ) and is recruited en bloc from the cytosol onto Golgi membranes. In mammals and yeast, α- and β'-COP WD40 domains mediate cargo-selective interactions with dilysine motifs present in canonical cargoes of COPI vesicles. In contrast to mammals and yeast, three isoforms of β'-COP (β'1-3-COP) have been identified in Arabidopsis . To understand the role of Arabidopsis β'-COP isoforms in plant biology, we have identified and characterized loss-of-function mutants of the three isoforms, and double mutants were also generated. We have found that the trafficking of a canonical dilysine cargo (the p24 family protein p24δ5) is affected in β'-COP double mutants. By western blot analysis, it is also shown that protein levels of α-COP are reduced in the β'-COP double mutants. Although none of the single mutants showed an obvious growth defect, double mutants showed different growth phenotypes. The double mutant analysis suggests that, under standard growth conditions, β'1-COP can compensate for the loss of both β'2-COP and β'3-COP and may have a prominent role during seedling development.

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis ). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1 / PGAP3 like genes ( AtPGAP3A and AtPGAP3B ). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bernat-Silvestre, Ma, Johnson, Ferrando, Aniento and Marcote.)

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signaling, and cell wall biosynthesis. The GPI anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodeled. In mammals and yeast, this remodeling is required for GPI-APs to be included in Coat Protein II-coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodeling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and Post-GPI Attachment to Proteins Inositol Deacylase 1 (PGAP1, mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis (Arabidopsis thaliana) PGAP1 localizes to the ER and likely functions as the GPI inositol-deacylase that cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Coat Protein I (COPI) consists of a complex (coatomer) formed by seven subunits (α-, β-, β'-, γ-, δ-, ε-, and ζ-COP) that is recruited to Golgi membranes to form vesicles that shuttle from the Golgi apparatus to the ER and between Golgi stacks. Recently, it has been described that loss of function mutants of the two Arabidopsis β-COP genes, β1-COP and β2-COP, showed increased sensitivity to salt stress (NaCl). Using a mixture of either Na + or Cl - salts, we have now found that β-COP mutants are specifically and highly sensitive to chloride ions.

Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for different subcellular compartments. This protocol describes a transient transformation assay with five-day-old seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows high-throughput screening of new constructs and the study of the localization of a large number of subcellular markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.

p24 proteins are a family of type-I membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi apparatus via Coat Protein I (COPI)- and COPII-coated vesicles. These proteins have been proposed to function as cargo receptors, but the identity of putative cargos in plants is still elusive. We previously generated an Arabidopsis ( Arabidopsis thaliana ) quadruple loss-of-function mutant affecting p24 genes from the δ-1 subclass of the p24 delta subfamily ( p24δ3δ4δ5δ6 mutant). This mutant also had reduced protein levels of other p24 family proteins and was found to be sensitive to salt stress. Here, we used this mutant to test the possible involvement of p24 proteins in the transport to the plasma membrane of glycosylphosphatidylinositol (GPI)-anchored proteins. We found that GPI-anchored proteins mostly localized to the ER in p24δ3δ4δ5δ6 mutant cells, in contrast to plasma membrane proteins with other types of membrane attachment. The plasma membrane localization of GPI-anchored proteins was restored in the p24δ3δ4δ5δ6 mutant upon transient expression of a single member of the p24 δ-1 subclass, RFP-p24δ5, which was dependent on the coiled-coil domain in p24δ5. The coiled-coil domain was also important for a direct interaction between p24δ5 and the GPI-anchored protein arabinogalactan protein4 (AGP4). These results suggest that Arabidopsis p24 proteins are involved in ER export and transport to the plasma membrane of GPI-anchored proteins., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

The early secretory pathway involves bidirectional transport between the endoplasmic reticulum (ER) and the Golgi apparatus and is mediated by coat protein complex I (COPI)-coated and coat protein complex II (COPII)-coated vesicles. COPII vesicles are involved in ER to Golgi transport meanwhile COPI vesicles mediate intra-Golgi transport and retrograde transport from the Golgi apparatus to the ER. The key component of COPI vesicles is the coatomer complex, that is composed of seven subunits (α/β/β'/γ/δ/ε/ζ). In Arabidopsis two genes coding for the β-COP subunit have been identified, which are the result of recent tandem duplication. Here we have used a loss-of-function approach to study the function of β-COP. The results we have obtained suggest that β-COP is required for plant growth and salt tolerance. In addition, β-COP function seems to be required for maintaining the structure of the Golgi apparatus., (Copyright © 2020 Sánchez-Simarro, Bernat-Silvestre, Gimeno-Ferrer, Selvi-Martínez, Montero-Pau, Aniento and Marcote.)

The p24 family of proteins (also known as the TMED family) are key regulators of protein trafficking along the secretory pathway, but very little is known about their functions in plants. A quadruple loss-of-function mutant affecting the p24 genes from the δ-1 subclass of the p24δ subfamily ( p24δ3δ4δ5δ6 ) showed alterations in the Golgi, suggesting that these p24 proteins play a role in the organization of the compartments of the early secretory pathway in Arabidopsis Loss of p24δ-1 proteins also induced the accumulation of the K/HDEL receptor ERD2a (ER lumen protein-retaining receptor A) at the Golgi and increased secretion of BiP family proteins, ER chaperones containing an HDEL signal, probably due to an inhibition of COPI-dependent Golgi-to-ER transport of ERD2a and thus retrieval of K/HDEL ligands. Although the p24δ3δ4δ5δ6 mutant showed enhanced sensitivity to salt stress, it did not show obvious phenotypic alterations under standard growth conditions. Interestingly, this mutant showed a constitutive activation of the unfolded protein response (UPR) and the transcriptional upregulation of the COPII subunit gene SEC31A , which may help the plant to cope with the transport defects seen in the absence of p24 proteins., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The K/HDEL receptor ERD2 mediates the transport of soluble endoplasmic reticulum (ER)-resident proteins containing a C-terminal K/HDEL signal from the Golgi apparatus back to the ER via COPI (COat Protein I)-coated vesicles. Sorting of ERD2 within COPI vesicles is facilitated by p24 proteins. In Arabidopsis, p24δ5 has been shown to interact directly with ERD2 via its luminal GOLD (GOLgi Dynamics) domain and with COPI proteins via its cytoplasmic C-terminal tail at the acidic pH of the Golgi apparatus. Several members of the p24 family in mammals and yeast have been shown to be glycosylated, but whether Arabidopsis p24 proteins are glycosylated and the role of the sugar moiety in p24 function remain unclear. Here, we show that Arabidopsis p24δ5 protein is N-glycosylated in its GOLD domain. Furthermore, we demonstrate that this post-translational modification is important for its coupled transport with p24β2 at the ER-Golgi interface, for its interaction with the K/HDEL receptor ERD2, and for retrograde transport of ERD2 and K/HDEL ligands from the Golgi apparatus back to the ER., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β'-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

PIN-FORMED (PIN) family proteins direct polar auxin transport based on their asymmetric (polar) localization at the plasma membrane. In the case of PIN1, it mainly localizes to the basal (rootward) plasma membrane domain of stele cells in root meristems. Vesicular trafficking events, such as clathrin-dependent PIN1 endocytosis and polar recycling, are probably the main determinants for PIN1 polar localization. However, very little is known about the signals which may be involved in binding the μ-adaptin subunit of clathrin adaptor complexes (APs) for sorting of PIN1 within clathrin-coated vesicles, which can determine its trafficking and localization. We have performed a systematic mutagenesis analysis to investigate putative sorting motifs in the hydrophilic loop of PIN1. We have found that a non-canonical motif, based in a phenylalanine residue, through the binding of μA(μ2)- and μD(μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 traffcking along the secretory pathway, respectively. In addition, tyrosine-based motifs, which also bind different μ-adaptins, could also contribute to PIN1 trafficking and localization.

p24 family proteins have been known for a long time, but their functions have remained elusive. However, they are emerging as essential regulators of protein trafficking along the secretory pathway, influencing the composition, structure, and function of different organelles in the pathway, especially the ER and the Golgi apparatus. In addition, they appear to modulate the transport of specific cargos, including GPI-anchored proteins, G-protein-coupled receptors, or K/HDEL ligands. As a consequence, they have been shown to play specific roles in signaling, development, insulin secretion, and the pathogenesis of Alzheimer's disease. The search of new putative ligands may open the way to discover new functions for this fascinating family of proteins.

In contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a μA (μ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a μD (μ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of μA (μ2)- and μD (μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

The p24 proteins belong to a family of type I membrane proteins which cycle between the endoplasmic reticulum (ER) and Golgi via coat protein I (COPI) and COPII vesicles. Current nomenclature classifies them into four subfamilies, although plant p24 proteins belong to either the p24β or the p24δ subfamilies. Here, we show that Arabidopsis p24δ5/δ9 and HDEL ligands shift the steady-state distribution of the K/HDEL receptor ERD2 from the Golgi to the ER. We also show that p24δ5/δ9 interact directly with ERD2. This interaction requires the Golgi dynamics (GOLD) domain in p24δ5 and is much higher at acidic than at neutral pH, consistent with both proteins interacting at the cis-Golgi. In addition, p24δ5 also inhibits the secretion of HDEL ligands, but not constitutive secretion, showing a role for p24δ5 in retrograde Golgi-to-ER transport. Both p24δ5 and ERD2 interact with ADP-ribosylation factor 1 (ARF1) and COPI subunits, mostly at acidic pH, consistent with COPI vesicles being involved in retrograde transport of both proteins. In contrast, both proteins interact with the COPII subunit Sec23, mostly at neutral pH, consistent with this interaction taking place at the ER for anterograde transport to the Golgi apparatus., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24β3 mainly localizes to the Golgi apparatus (as p24β2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24β3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the β and δ subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 localizes to the endoplasmic reticulum (ER) as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. Using specific antibodies, endogenous p24δ5 has now been localized to the ER and p24β2 to the Golgi apparatus in Arabidopsis root tip cells by immunogold electron microscopy. The relative contributions of the cytosolic tail and the luminal domains to p24δ5 trafficking have also been characterized. It is demonstrated that whereas the dilysine motif in the cytoplasmic tail determines the location of p24δ5 in the early secretory pathway, the luminal domain may contribute to its distribution downstream of the Golgi apparatus. By using knock-out mutants and co-immunoprecipitation experiments, it is shown that p24δ5 and p24β2 interact with each other. Finally, it is shown that p24δ5 and p24β2 exhibit coupled trafficking at the ER-Golgi interface. It is proposed that p24δ5 and p24β2 interact with each other at ER export sites for ER exit and coupled transport to the Golgi apparatus. Once in the Golgi, p24δ5 interacts very efficiently with the COPI machinery for retrograde transport back to the ER.

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

p24 proteins constitute a family of putative cargo receptors that traffic in the early secretory pathway. p24 proteins can be divided into four subfamilies (p23, p24, p25 and p26) by sequence homology. In contrast to mammals and yeast, most plant p24 proteins contain in their cytosolic C-terminus both a dilysine motif in the -3, -4 position and a diaromatic motif in the -7, -8 position. We have previously shown that the cytosolic tail of Arabidopsis p24 proteins has the ability to interact with ARF1 and coatomer (through the dilysine motif) and with COPII subunits (through the diaromatic motif). Here, we establish the localization and trafficking properties of an Arabidopsis thaliana p24 protein (Atp24) and have investigated the contribution of the sorting motifs in its cytosolic tail to its in vivo localization. Atp24-red fluorescent protein localizes exclusively to the endoplasmic reticulum (ER), in contrast with the localization of p24 proteins in other eukaryotes, and the dilysine motif is necessary and sufficient for ER localization. In contrast, Atp24 mutants lacking the dilysine motif are transported along the secretory pathway to the prevacuolar compartment and the vacuole, although a significant fraction is also found at the plasma membrane. Finally, we have found that ER export of Atp24 is COPII dependent, while its ER localization requires COPI function, presumably for efficient Golgi to ER recycling.

Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in plants. In Arabidopsis, 20 MAPKs have been identified and classified into four major groups (A-D). Little is known about the role of group C MAPKs. We have studied the activation of Arabidopsis subgroup C1 MAPKs (AtMPK1/AtMPK2) in response to mechanical injury. An increase in their kinase activity was detected in response to wounding that was blocked by cycloheximide. Jasmonic acid (JA) activated AtMPK1/AtMPK2 in the absence of wounding. Wound and JA-induction of AtMPK1/2 kinase activity was not prevented in the JA-insensitive coi1 mutant. Other stress signals, such as abscisic acid (ABA) and hydrogen peroxide, activated AtMPK1/2. This report shows for the first time that regulation of AtMPK1/2 kinase activity in Arabidopsis might be under the control of signals involved in different kinds of stress.

Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits the interaction between the YTRF endocytosis signal in the hTfR cytosolic tail and the mu2-subunit of the AP2 complex. Indeed, tyrphostin A23 inhibits Tfn internalization and redistributes most of hTfR to the plasma membrane, suggesting that the endocytosis signal of hTfR is functional in Arabidopsis protoplasts. Co-immunoprecipitation experiments show that hTfR is able to interact with a mu-adaptin subunit from Arabidopsis cytosol, a process that is blocked by tyrphostin A23. In contrast, treatment with brefeldin A, which inhibits recycling from endosomes back to the plasma membrane in plant cells, leads to the accumulation of Tfn and hTfR in larger patches inside the cell, reminiscent of BFA compartments. Therefore, hTfR has the same trafficking properties in Arabidopsis protoplasts as in animal cells, and cycles between the plasma membrane and endosomal compartments. The specific inhibition of Tfn/hTfR internalization and recycling by tyrphostin A23 and BFA, respectively, thus provide valuable molecular tools to characterize RME and the recycling pathway in plant cells.

Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment.

PsMAPK3, a new MAP kinase cDNA, was cloned from ovaries of Pisum sativum L. Expression of PsMAPK3 is at low basal levels in unpollinated ovaries but it is rapidly induced by gibberellic acid (peak at 30 min) and 6-benzyladenine (peak at 45 min). Both treatments promoted the development of a parthenocarpic fruit. In situ hybridization localized PsMAPK3 mRNA in ovules. The transcript was additionally detected in the mesocarp when it is expanding in response to the treatments. These observations suggest that gibberellins and cytokinins regulate PsMAPK3 mRNA levels in pea ovary shortly after fruit set is induced.

Polyamines induce the transport in vitro of the precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria by facilitating its functional binding to these organelles. Comparative studies of the effect on the in vitro transport of pOTC of polyamine derivatives and related compounds have allowed us to establish that: (i) at least two protonated amino groups per molecule are necessary to induce the pOTC transport; (ii) a distance of three -CH2- groups between the amino groups in diamines is enough to induce this effect, although no differences were observed with diamines having distances of three to eight -CH2- groups. Longer distances resulted in a marked decrease of the effect.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.

A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from G0/G1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in G1.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.

A monodimensional thin layer chromatography method to separate several sugars of clinical interest is described. The separation and identification of 14 sugars (L-fucose, D-galactose, D-glucose, lactose, N-acetylglucosamine, D-maltose, D-manose, L-sorbase, fructose, D-xylose, glucuronic acid, N-acetyllactosamine, 3' and 6' sialyllactose) and maltodextrines (G(2)-G(8)) is possible by using two different eluents mixtures, as well as two different detection reagents. The method has been applied to separate sugars, maltodextrines and oligosaccharides in several biological fluids (blood, urine and faeces), in an infant milk and in human milk. It is a very simple technique (with a high sensitivity) that can be used in any lab.

Activation of the cdc2 kinase in the cell cycle occurs upon binding to a regulatory subunit called cyclin. Cyclin A associates with both Cdc2 and its homologue Cdk2. The two complexes appear in S phase but cyclin A/Cdk2 is activated earlier than cyclin A/Cdc2. Several regions in Cdc2 are involved in binding cyclins A and B. Phosphorylation of cyclin/Cdk complexes ensures that the kinase activity peaks at a specific time in the cell cycle. Phosphorylation of Thr161 in Cdc2 is required for strong cyclin binding and kinase activity in vitro; its dephosphorylation is necessary for cells to exit mitosis. We have identified a novel 'Activating factor' that stimulates binding between cyclin and Cdc2 by inducing phosphorylation of Cdc2 on Thr161. We propose that Thr161 is targeted by an additional cell cycle regulatory pathway.

Polyamines induce the transport in vitro of the rat liver precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria. The accumulation of this precursor at the level of binding to the mitochondrial surface has allowed us to establish that polyamines are involved in the interaction of the precursor with the mitochondrial surface. Transport of a chimeric protein having the signal sequence of pOTC fused to a fragment of the cytosolic protein human arginosuccinate lyase was also induced by polyamines. The sensitivity of the pOTC synthesized in vitro and of the chimeric protein to proteinases decreases in the presence of polyamines. This result suggests that polyamines may play a role in modulating the folding of precursors to favour their binding to mitochondria.

Rat liver mitochondria were loaded with cytochrome c by incubation with large amounts of [14C]apocytochrome c. After being washed they were incubated with either more apocytochrome c or cytochrome c. There was no release of labeled proteins from the mitochondria when incubated with cytochrome c. However, there was when incubated with apocytochrome c. The material released showed only one radioactive band which migrated as cytochrome c. Also no release of proteins other than cytochrome c was detected when liver mitochondria isolated from rats injected with [35S]methionine were incubated with apocytochrome c. These results suggest that the level and possibly the turnover of cytochrome c in rat liver mitochondria is regulated by the entry of apocytochrome c into mitochondria.

The polyamines spermidine, spermine and putrescine, by themselves, at physiological concentrations, induce the transport of the precursor of ornithine carbamoyltransferase into isolated rat liver mitochondria. The presence of polyamines in the transport medium results in the approach of both mitochondrial membranes, suggesting a possible role of these molecules in the transport of the precursor of ornithine carbamoyltransferase into mitochondria, by the formation and/or stabilization of mitochondrial structures involved in the transport system.

The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.

To establish the interrelationship between protein synthesis and degradation, we have developed a simple procedure. We have chosen the mitochondrial proteins, because most of them are synthesized outside mitochondria and are then imported into the organelle. This fact allows to separate easily the synthesis steps from the general turnover. By using this system we have some evidences suggesting that the in vitro protein concentration in mitochondria may be regulated by the entry of mitochondrial protein precursors. Also, we are studying the factors that regulate the import of mitochondrial protein precursors into the organelle, because this step may be essential in the regulation of the turnover of mitochondrial proteins.