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The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31. Show less

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking. Show less

Cereal embryos sustain severe water deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. MS analysis allowed the identification of 28 proteins involved in stress tolerance (late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that late embryogenesis abundant Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance. Show less

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs. Show less

There are few efficient promoters for use with stress-inducible gene expression in plants, and in particular for monocotyledonous crops. Here, we report the identification of six genes, Rab21, Wsi18, Lea3, Uge1, Dip1, and R1G1B that were induced by drought stress in rice microarray experiments. Gene promoters were linked to the gfp reporter and their activities were analyzed in transgenic rice plants throughout all stages of plant growth, from dry seeds to vegetative tissues to flowers, both before and after drought treatments. In fold induction levels, Rab21 and Wsi18 promoters ranged from 65- and 36-fold in leaves to 1,355- and 492-fold in flowers, respectively, whereas Lea3 and Uge1 were higher in leaves, but lower in roots and flowers, as compared with Rab21 and Wsi18. Dip1 and R1G1B promoters had higher basal levels of activity under normal growth conditions in all tissues, resulting in smaller fold-induction levels than those of the others. In drought treatment time course, activities of Dip1 and R1G1B promoters rapidly increased, peaked at 2 h, and remained constant until 8 h, while that of Lea3 slowly yet steadily increased until 8 h. Interestingly, Rab21 activity increased rapidly and steadily in response to drought stress until expression peaked at 8 h. Thus, we have isolated and characterized six rice promoters that are all distinct in fold induction, tissue specificity, and induction kinetics under drought conditions, providing a variety of drought-inducible promoters for crop biotechnology. Show less

Carcinoma-associated fibroblasts (CAFs) can promote the progression of tumours in many ways. They can remodel the extracellular matrix to generate an environment that enables the invasion of cancer cells. We hypothesised that compounds that prevent matrix remodelling by CAFs would block their ability to promote carcinoma cell invasion. We designed a screen for compounds that interfere with CAF-promoted matrix remodelling. Hits from this screen were investigated in organotypic invasion models of squamous cell carcinoma (SCC). We find that lovastatin and simvastatin reduce matrix remodelling by fibroblasts and thereby reduce SCC invasion. This class of compounds exert their effects partly through disrupting the function of Rab proteins, and we show a new role for Rab21 in promoting cancer cell invasion promoted by CAFs. Rab21 is required for CAFs to promote the invasion of cancer cells. It enables the accumulation of integrin alpha5 at the plasma membrane and subsequent force-mediated matrix remodelling. Show less

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P(2) and PI(3,4,5)P(3) levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments. Show less

The Rab family of small GTPases functions in regulating vesicular transport in all eukaryotes. In the past few years, several important reports have linked some members of the Rab family to intriguing mechanistic aspects of cancer cell migration and invasiveness. Rab5 and Rab21 associate with alpha-integrin subunits and modulate their endosomal traffic and subcellular localization. Expression of the latter enhances adhesion and migration of certain cancer cell types. Rab25 has been functionally linked to tumor progression and the invasiveness of some epithelial cancers. Rab25 promotes invasive migration of cells in three-dimensional microenvironments by associating with alpha5beta1 integrin, and directing its recycling to dynamic ruffling protrusions at the migrating cell front. Acting directly, or through its effector, the Rab-coupling protein, Rab25 could potentially engage both integrin and epidermal growth factor receptor and enhance their oncogenic recycling and signaling. Tumor invasiveness may also be modulated by Rab8-mediated exocytic traffic of MT1-matrix metalloproteinase, with the latter's activity likely influenced by interaction with the mammalian suppressor of Sec4 (Mss4), a Rab8 guanine nucleotide exchange factor, and integrin. We discuss highlights in the recent literature that point towards a role for Rab-mediated membrane traffic in cancer cell migration and invasion. Show less

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes. Show less

The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP. Show less

Forster resonance energy transfer (FRET) is a powerful tool in studying biomolecular interactions. Intrinsically fluorescent lanthanide chelates are increasingly used as FRET donors due to their long emission lifetime that enables the use of time resolution. Fluorescent proteins, on the other hand, owe their popularity to the intrinsic luminescent properties, facilitating their use as fusion proteins. In this investigation, two energy transfer pairs, terbium(III) chelate with green fluorescent protein (GFP) and europium(III) chelate with yellow fluorescent protein (YFP), were studied by expressing the fluorescent protein acceptor as a fusion protein together with Rab21 GTPase. GTP-conjugated lanthanide chelates were used as donor conjugates. In contrast to conventional FRET observed with the Tb(3+)-GFP pair, a phenomenon called nonoverlapping FRET (nFRET) was observed with the Eu(3+)-YFP pair. In nFRET, the sensitized emission of the acceptor was measured at shorter wavelength than where the emission of the donor was observed. Regardless of the lower signal levels, nFRET resulted in a substantially higher signal-to-background ratio. Conventional FRET from sensitized acceptor yielded a single apparent fluorescence emission lifetime, while with nFRET two lifetimes were observed. The lanthanide chelates together with fluorescent proteins enable a straightforward and sensitive assay technology in nFRET applications. Show less

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer. Show less

Varp, a novel protein containing a VPS9 domain and ankyrin repeats, can function as a guanine nucleotide exchange factor (GEF) of Rab21. We previously reported that Varp plays an important role in the regulation of endosome dynamics. To further investigate the function of Varp, a yeast two-hybrid screen was performed and Rab38 was identified as a Varp-associated protein. We demonstrate that Varp physically interacts with Rab38, and preferentially binds to the active GTP-bound form of Rab38 both in vitro and in vivo. Furthermore, Varp was shown to be recruited to Rab38-positive organelles in an ankyrin-repeat 1 (ANK1)-dependent manner. Our data demonstrate that Varp is a potential effector of Rab38. Together with our previous study, we propose Varp serves as both an effector and a GEF by interacting with different Rabs in mammalian cells. Show less

We found that wortmannin, a potent phosphoinositide 3-kinase (PI3K) inhibitor, markedly induced the formation of Rab21-positive tubular compartments in A431 cells. By time-lapse fluorescence microscopy of live cells co-expressing fluorescent protein-fused Rab21 and other marker proteins, it was shown that the Rab21-positive tubules in wortmannin-treated cells were derived from Rab5-positive early endosomes, but not from late endosomes, recycling endosomes, lysosomes or the trans-Golgi network. The formation of Rab21-positive tubules was very dynamic and required microtubules. Rab21-positive tubules were also formed by the treatment of cells with 3-methyladenine (3-MA), which inhibits class III PI3K rather than class I PI3K. Furthermore, the loss of PI(3)P correlated with the tubulation of Rab21-positive endosomes in cells co-expressing fluorescent protein-fused Rab21 and a tandem FYVE domain. These results suggest that the lowering of PI(3)P as a result of class III PI3K inhibition may be an important cue for the morphological change of Rab21-positive early endosomes from vesicular to tubular form. Show less

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses. Show less

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR-PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs beta-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases. Show less

To identify gene expression patterns and interaction networks related to BCR-ABL status and clinical outcome in adults with acute lymphoblastic leukemia (ALL). DNA microarrays were used to profile a set of 54 adult ALL specimens from the Medical Research Council UKALL XII/Eastern Cooperative Oncology Group E2993 trial (21 p185BCR-ABL-positive, 16 p210BCR-ABL-positive and 17 BCR-ABL-negative specimens). Using supervised and unsupervised analysis tools, we detected significant transcriptomic changes in BCR-ABL-positive versus -negative specimens, and assessed their validity in an independent cohort of 128 adult ALL specimens. This set of 271 differentially expressed genes (including GAB1, CIITA, XBP1, CD83, SERPINB9, PTP4A3, NOV, LOX, CTNND1, BAALC, and RAB21) is enriched for genes involved in cell death, cellular growth and proliferation, and hematologic system development and function. Network analysis demonstrated complex interaction patterns of these genes, and identified FYN and IL15 as the hubs of the top-scoring network. Within the BCR-ABL-positive subgroups, we identified genes overexpressed (PILRB, STS-1, SPRY1) or underexpressed (TSPAN16, ADAMTSL4) in p185BCR-ABL-positive ALL relative to p210BCR-ABL-positive ALL. Finally, we constructed a gene expression- and interaction-based outcome predictor consisting of 27 genes (including GRB2, GAB1, GLI1, IRS1, RUNX2, and SPP1), which correlated with overall survival in BCR-ABL-positive adult ALL (P = .0001), independent of age (P = .25) and WBC count at presentation (P = .003). We identified prominent molecular features of BCR-ABL-positive adult ALL, which may be useful for developing novel therapeutic targets and prognostic markers in this disease. Show less

VARP is a novel VPS9 domain-containing protein which acts as a guanine nucleotide exchange factor for small GTPases Rab21 and Rab5, regulators of early endocytosis. However, the molecular mechanisms underlying VARP activity regulation and intracellular localization remain unknown. Using protein interaction cloning in yeast we isolated multiadaptor proteins of 4.1 protein family and RanBP9 as putative VARP interaction partners. The interactions revealed might be important for proper intracellular localization of VARP and its functions in early endocytosis. Show less

RABEX-5 and other exchange factors with VPS9 domains regulate endocytic trafficking through activation of the Rab family GTPases RAB5, RAB21 and RAB22. Here we report the crystal structure of the RABEX-5 catalytic core in complex with nucleotide-free RAB21, a key intermediate in the exchange reaction pathway. The structure reveals how VPS9 domain exchange factors recognize Rab GTPase substrates, accelerate GDP release and stabilize the nucleotide-free conformation. We further identify an autoinhibitory element in a predicted amphipathic helix located near the C terminus of the VPS9 domain. The autoinhibitory element overlaps with the binding site for the multivalent effector RABAPTIN-5 and potently suppresses the exchange activity of RABEX-5. Autoinhibition can be partially reversed by mutation of conserved residues on the nonpolar face of the predicted amphipathic helix or by assembly of the complex with RABAPTIN-5. Show less

The small GTPases Rab5 and Rab21 are closely related, and play essential roles in endocytic trafficking. Rab5 is regulated by VPS9-domain-containing guanine nucleotide exchange factors. Here, we describe a new VPS9-domain protein with ankyrin repeats, the VPS9-ankyrin-repeat protein (Varp). Varp interacts preferentially with GDP-bound Rab21 and has a much stronger guanine nucleotide exchange activity towards Rab21 than Rab5. Furthermore, RNAi-mediated depletion of endogenous Varp significantly disrupts the activity of Rab21 in HeLa cells. Ectopically expressed Varp mainly localizes to early endosomes and causes enlargement of early endosomes and giant late endosomes. Both the VPS9 domain and ankyrin-repeats are required for the endosomal localization and the activity of Varp in vivo. These results suggest that Varp is a potential Rab21 guanine nucleotide exchange factor and might regulate endosome dynamics in vivo. Show less

Dynamic turnover of integrin cell adhesion molecules to and from the cell surface is central to cell migration. We report for the first time an association between integrins and Rab proteins, which are small GTPases involved in the traffic of endocytotic vesicles. Rab21 (and Rab5) associate with the cytoplasmic domains of alpha-integrin chains, and their expression influences the endo/exocytic traffic of integrins. This function of Rab21 is dependent on its GTP/GDP cycle and proper membrane targeting. Knock down of Rab21 impairs integrin-mediated cell adhesion and motility, whereas its overexpression stimulates cell migration and cancer cell adhesion to collagen and human bone. Finally, overexpression of Rab21 fails to induce cell adhesion via an integrin point mutant deficient in Rab21 association. These data provide mechanistic insight into how integrins are targeted to intracellular compartments and how their traffic regulates cell adhesion. Show less

Endocytic pathways are highly dynamic gateways for molecules to enter cells. Functionality and specificity is in part controlled by a number of small GTPases called Rabs. In defined cellular locations, Rabs mediate multiple functions in membrane trafficking via their specific interaction with organelle membranes and a host of affector and effector molecules. On endocytic pathways, Rabs have been shown to control the formation of vesicles on the plasma membrane and the downstream delivery of internalized molecules to a number of cellular locations. As numerous Rabs are located to endocytic pathways, an internalized molecule may traverse a number of Rab specific substations or subdomains en route to its final destination. Rabs 5, 21 and 22 have all been localized to the early endocytic pathway and have been shown to share a number of characteristics to merit their segregation into a single functional endocytic group. In this review, we compare experiments that describe similarities and differences in endosome morphology and function that is mediated by their expression in cells. Show less

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity. Show less

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics. Show less

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells. Show less

To explore the potential role of Rab GTPases in human insulin resistance, we first employed a PCR-cloning approach to identify Rab isoforms that are expressed in human skeletal muscle. Multiple Rab isoforms including Rab1A, Rab4A, Rab5B, Rab7, Rab8, Rab10, Rab12A, Rab13, Rab18, Rab21, and Rab22 mRNA were found to be expressed in human skeletal muscle. The second goal was to examine whether mRNA expression for Rabs targeted to endocytotic/exocytotic compartments was altered as a function of insulin resistance. Quantitative PCR analysis demonstrated that Rab4A, Rab5B and Rab18 mRNA levels in skeletal muscle from insulin-resistant patients without (IR) and with non-insulin-dependent diabetes mellitus (NIDDM) were not significantly different from those in insulin-sensitive controls (IS). At the protein level, total Rab5B amount was not significantly different among IS, IR and NIDDM subgroups. However, in basal muscle, Rab5B in the total membrane fraction was 2.1-3.6 fold higher in IR and NIDDM than in IS subjects. Insulin increased membrane-associated Rab5B by 3-fold in IS subjects, whereas this effect was not significant in both IR and NIDDM subgroups. Thus, for the first time, we have comprehensively studied the mRNA expression of Rab isoforms in human muscle. The phlethora of Rab GTPases are indicative of high volume of vesicular traffic and regulated metabolism. The potential role of specific Rab isoforms in insulin resistance does not rely on a change in steady state mRNA levels, but is demonstrable as an alteration in protein subcellular distribution and trafficking. Show less

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell. Show less

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes. Show less

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos. Show less

The effects of cold on protein and RNA metabolism in leaves of rice (Oryza sativa L.) seedlings were investigated. Treatment with a diurnal cycle of 15/10 degrees C or 11/6 degrees C for up to 1 week resulted in progressive changes in the protein synthesis pattern after in vivo labeling of intact rice leaves with [(35)S]methionine. These changes were reversed when the seedlings were returned to normal growth temperatures. While de novo accumulation of several abundant proteins was suppressed, some polypeptides were consistently found to be cold-induced. Synthesis of ribulose 1,5-bisphosphate carboxylase (Rubisco) was drastically reduced after 7 days of cold. Using immunoprecipitation of Rubisco, evidence was obtained that the suppression was greater for the small subunit (over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their synthesis. Preformed Rubisco as well as other cold-suppressed proteins were stable for up to 7 days at 11/6 degrees C. Cold-sensitive rice cultivars responded with similar but more drastic changes in the protein synthesis pattern when compared to cold-tolerant varieties. The suppression of Rubisco synthesis by cold was shown to result from reduced levels of the mRNAs encoding both subunits; their decrease paralleled the lower protein synthesis of each. The levels of other chloroplast-encoded mRNAs, especially psaB, and of the nuclear encoded chlorophyll a/b binding protein, also strongly decreased in the cold, whereas the transcripts of the mitochondrial genes apt9, coxIII, and most nuclear genes analyzed were unaffected or only slightly reduced. These data indicate that some chloroplast functions are disturbed during cold stress. One nuclear gene known to be induced by water stress and ABA (Rab21) was also found to be induced by cold treatment. Show less