Cloning and Characterization of ApRab4, a Recycling Rab Protein of Aiptasia pulchella, and Its Implication in the Symbiosome Biogenesis

The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin–avidin detection system, we found evidence for symbiosome–endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome.     

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3f^T22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.     

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.     

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.     

Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.     

Engagement of the Small GTPase Rab31 Protein and Its Effector,Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.     

RabGEFs are a major determinant for specific Rab membrane targeting

Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.     

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.     

八肋游仆虫Rab家族基因克隆和多样性分析

Rab蛋白是在真核细胞内膜泡运输过程中起重要调节作用的一类小分子Ras-like蛋白,为Ras超家族中最大的家族。Rab家族成员在不同的生物中表现出数量的多样性和功能上的分化。为进一步了解Rab蛋白的多样性及其在真核细胞内膜泡运输网络中的功能,本研究利用游仆虫大核染色体特异的端粒结构和基因大小的染色体结构特征,通过简并引物PCR方法从八肋游仆虫(Euplotes octocarinatus)中克隆到9种新的Rab基因,分别为EoRab1A、EoRab2b、EoRab2c、EoRab2d、EoRab6、EoRab7、EoRab2-like、EoRabL2和EoRan(GenBank登陆号为HM371131～HM371139)。序列分析表明,游仆虫中Rab基因家族成员既包括具有维持细胞结构核心功能保守基因,又包括为适应环境而进化出的特殊功能的新基因。     

Endocytic membrane trafficking in the control of centrosome function

Until recently, endocytic trafficking and its regulators were thought to function almost exclusively on membrane-bound organelles and/or vesicles containing a lipid bilayer. Recent studies have demonstrated that endocytic regulatory proteins play much wider roles in trafficking regulation and influence a variety of nonendocytic pathways, including trafficking to/from mitochondria and peroxisomes. Moreover, new studies also suggest that endocytic regulators also control trafficking to and from cellular organelles that lack membranes, such as the centrosome. Although endocytic membrane trafficking (EMT) clearly impacts pathways downstream of the centrosome, such as ciliogenesis (including transport to and from cilia), mitotic spindle formation, and cytokinesis, relatively few studies have focused on the growing role for EMT more directly on centrosome biogenesis, maintenance and control throughout cell cycle, and centrosome duplication. Indeed, a growing number of endocytic regulatory proteins have been implicated in centrosome regulation, including various Rab proteins (among them Rab11) and the leucine-rich repeat kinase 2. In this review, we will examine the relationship between centrosomes and EMT, focusing primarily on how EMT directly influences the centrosome.     

Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells

Heregulin beta1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38(MAPK) and p42/44(MAPK). Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma.     

ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the Aiptasia-Symbiodinium endosymbiosis

Endosymbiotic association of the Symbiodinium dinoflagellates (zooxanthellae) with their cnidarian host cells involves an alteration in the development of the alga-enclosing phagosomes. To uncover its molecular basis, we previously investigated and established that the intracellular persistence of the zooxanthella-containing phagosomes involves specific alga-mediated interference with the expression of ApRab5 and ApRab7, two key endocytic regulatory Rab proteins, which results in the selective retention of the former on and exclusion of the later from the organelles. Here we examined the role of ApRab11, a cnidarian homologue of the key endocytic recycling regulator, Rab11, in the Aiptasia-Symbiodinium endosymbiosis. ApRab11 protein shared 88% overall sequence identity with human Rab11A and contained all Rab-specific signature motifs. Co-localization and mutagenesis studies showed that EGFP-tagged ApRab11 was predominantly associated with recycling endosomes and functioned in the recycling of internalized transferrin. In phagocytosis of latex beads, ApRab11 was quickly recruited to and later gradually removed from the developing phagosomes. Significantly, although ApRab11 immunoreactivity was rapidly detected on the phagosomes containing either newly internalized, heat-killed zooxanthellae, or resident zooxanthellae briefly treated with the photosynthesis inhibitor DCMU, it was rarely observed in the majority of phagosomes containing either newly internalized live, or healthy resident, zooxanthellae. It was concluded that through active exclusion of ApRab11 from the phagosomes in which they reside, zooxanthellae interfere with the normal recycling process required for efficient phagosome maturation, and thereby, secure their intracellular persistence, and consequently their endosymbiotic relationship with their cnidarian hosts.     

A Highlights from MBoC Selection: Rab21 in enterocytes participates in intestinal epithelium maintenance

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery. We focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, we identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. We further showed that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, we validated changes in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. Our data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.     

Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation,Guiding the Specific Vesicles to the Cyst Wall

Background

Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.

Methodology and Principal Findings

In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol.

Conclusions and Significance

Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.     

Rab3D regulates a novel vesicular trafficking pathway that is required for osteoclastic bone resorption

Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our data indicate that Rab3D is the major Rab3 species expressed in osteoclasts. To investigate the role of Rab3D in osteoclast physiology we examined the skeletal architecture of Rab3D-deficient mice and found an osteosclerotic phenotype. Although basal osteoclast number in null animals is normal the total eroded surface is significantly reduced, suggesting that the resorptive defect is due to attenuated osteoclast activity. Consistent with this hypothesis, ultrastructural analysis reveals that Rab3D(-/-) osteoclasts exhibit irregular ruffled borders. Furthermore, while overexpression of wild-type, constitutively active, or prenylation-deficient Rab3D has no significant effects, overexpression of GTP-binding-deficient Rab3D impairs bone resorption in vitro. Finally, subcellular localization studies reveal that, unlike wild-type or constitutively active Rab3D, which associate with a nonendosomal/lysosomal subset of post-trans-Golgi network (TGN) vesicles, inactive Rab3D localizes to the TGN and inhibits biogenesis of Rab3D-bearing vesicles. Collectively, our data suggest that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption.     

Crystal structure of inactive form of Rab3B

Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.     

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.     

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport,Alleviates Endoplasmic Reticulum Stress,and Reduces Lysosome Overload in Cystinosis

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns^−/− mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns^−/− cells. Ctns^−/− cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.     

Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.     

The C. elegans Rab Family: Identification,Classification and Toolkit Construction

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).