Genetic mapping of the mouse Rab7 gene and pseudogene and of the human RAB7 homolog

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5′ region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps. Received: 27 October 1997 / Accepted: 1 January 1998     

Rab27a and Rab27b Regulate Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by Independent Mechanisms

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a^ash/ash), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a^ash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a‐deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a^ash/ash and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27‐deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27‐deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.     

Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells

The four Rab3 paralogs A–D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD^?/?) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD^?/? animals large dense‐core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD^?/? cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short‐term (4–6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.     

Phosphorylation mediates the nuclear targeting of the maize Rab17 protein

The maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.     

The Rab Protein Family: Genetic Mapping of Six Rab Genes in the Mouse

Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an intersubspecific backcross [C57BL/6J-bg^J × (C57BL/6J-bg^J × CAST/Ei)F₁] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F₁ and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg , they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively.     

Bacterial Pathogens Commandeer Rab GTPases to Establish Intracellular Niches

Intracellular bacterial pathogens deploy virulence factors termed effectors to inhibit degradation by host cells and to establish intracellular niches where growth and differentiation take place. Here, we describe mechanisms by which human bacterial pathogens (including Chlamydiae; Coxiella burnetii; Helicobacter pylori; Legionella pneumophila; Listeria monocytogenes; Mycobacteria; Pseudomonas aeruginosa, Salmonella enterica) modulate endocytic and exocytic Rab GTPases in order to thrive in host cells. Host cell Rab GTPases are critical for intracellular transport following pathogen phagocytosis or endocytosis. At the molecular level bacterial effectors hijack Rab protein function to: evade degradation, direct transport to particular intracellular locations and monopolize host vesicles carrying molecules that are needed for a stable niche and/or bacterial growth and differentiation. Bacterial effectors may serve as specific receptors for Rab GTPases or as enzymes that post‐translationally modify Rab proteins or endosomal membrane lipids required for Rab function. Emerging data indicate that bacterial effector expression is temporally and spatially regulated and multiple virulence factors may act concertedly to usurp Rab GTPase function, alter signaling and ensure niche establishment and intracellular bacterial growth, making this field an exciting area for further study.      

Rab33b and Rab6 are Functionally Overlapping Regulators of Golgi Homeostasis and Trafficking

We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra‐Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra‐Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex‐ or Zeste White 10 (ZW10)‐depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP‐restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b‐dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra‐Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP‐Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga‐like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra‐Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra‐Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.     

Rab17‐mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion

Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.     

YAC Contigs of theRab1andwobbler(wr) Spinal Muscular Atrophy Gene Region on Proximal Mouse Chromosome 11 and of the Homologous Region on Human Chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized thewobblerspinal atrophy genewrto proximal mouse Chr 11, tightly linked toRab1,a gene coding for a small GTP-binding protein, andGlns-ps1,an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of theRab1region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescencein situhybridization (FISH), and sequence-tagged site (STS) isolation and mapping.Rab1andGlns-ps1were found to be only 200 kb apart. A potential CpG island near a methylatedNarI site and a trapped exon,ETG1.1,were found between these loci, and a new STS,AHY1.1,was found over 250 kb fromRab1.Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising theRAB1locus,AHY1.1,and the human homologue ofETG1.1,indicating a high degree of conservation of this region in the two species. We mappedAHY1.1and thus humanRAB1on Chr 2p13.4–p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the geneLMGMD2Bfor a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13–p16. The conservation between the mouseRab1and humanRAB1regions will be helpful in identifying candidate genes for thewobblerspinal muscular atrophy and in clarifying a possible relationship betweenwrandLMGMD2B.     

Rab18 binds PLIN2 and ACSL3 to mediate lipid droplet dynamics

Lipid droplet (LD) is a vital organelle governing lipid homeostasis and Rab18 has been linked to lipid metabolism. However, the mechanisms of Rab18-mediated LD dynamics in myoblast cells remain elusive. Here, we report that Rab18 plays an important role in oleic acid (OA)-induced LD accumulation in mouse myoblast C2C12 cells. Rab18 was translocated from the endoplasmic reticulum (ER) to LDs during LD accumulation, which was regulated by perilipin 2 (PLIN2), a major LD protein. LD-associated Rab18 bound with the C terminus of PLIN2 and the LD localization of Rab18 was diminished when PLIN2 was depleted. Moreover, loss of function of Rab18 led to reduced triacylglycerol (TAG) level and fewer but larger LDs. In contrast, overexpression of Rab18 resulted in elevated TAG content and LD number. Furthermore, LD-associated Rab18 interacted with acyl-CoA synthetase long-chain family member 3 (ACSL3), which in turn promoted the LD localization of this protein. These data show that Rab18 interacts with PLIN2 and forms a complex with PLIN2 and ACSL3, which plays a critical role in LD accumulation and dynamics of myoblast cells.     

TLR sorting by Rab11 endosomes maintains intestinal epithelial‐microbial homeostasis

Compartmentalization of Toll‐like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell‐intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild‐type controls, germ‐free Rab11a‐deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host‐microbial homeostasis at least partially via sorting TLRs.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

Rab6 functions in polarized transport in Drosophila photoreceptors

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected.

The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.     

Rab35 regulates neurite outgrowth and cell shape

Recent studies have identified Rab35 in the endocytic pathway and as a regulator of cytokinesis; however its molecular mechanisms are currently unknown. Here, we find that Rab35 colocalizes with actin filaments and with Cdc42, Rac1 and RhoA, and that Rab35 can activate Cdc42 both in vivo and in vitro. We find activated Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway and that siRNA knockdown of Rab35 activity abolishes neurite outgrowth in these cell lines. We conclude that one function of Rab35 is to regulate Rho-family GTPases and that this role has consequences for neurite outgrowth.

Structured summary

MINT-7012081: Rac1(uniprotkb:P63000) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012070: actin (uniprotkb:P60709) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012095: cdc42 (uniprotkb:P60953) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development

Developmental signalling pathways are regulated by intracellular vesicle trafficking in multicellular organisms. In our earlier communication, we have shown that mutation in Rab11 (a subfamily of the Ypt/Rab gene family) results in the activation of JNK signalling pathways in Drosophila eye. Here, we report that Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development. Using immunofluorescence and immunohistochemical analyses, we show that overexpression of Rab11 in mutant wing imaginal disc cells triggers the induction of apoptosis and activation of JNK and ERK. Further, using a genetic approach it has been shown that Rab11 interacts with the components of these pathways during Drosophila wing development. In addition to this, in Rab11 mutant wing imaginal discs JNK activity was monitored using puc^E69, a P‐lacZ enhancer‐trap line inserted in puckered (puc). A strong induction of puc in Rab11 mutant wing imaginal disc cells provided a strong support that Rab11 regulates the JNK signalling pathway during Drosophila wing development.     

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

A mutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype "chocolate" (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38(G19V) is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.     

Rab10 Regulates Phagosome Maturation and Its Overexpression Rescues Mycobacterium‐Containing Phagosomes Maturation

Phagosome maturation follows a defined biochemical program and, in the vast majority of cases, the microbe inside the phagosome is killed and digested. Although, an important number of pathogens, including Mycobacterium tuberculosis, which kills around two million people every year, have acquired the ability to survive, and even replicate by arresting phagosomal maturation. To identify more of the machinery involved in phagocytosis and phagosomal maturation, we investigated the function of Rab10 in engulfment and maturation of inert particles and Mycobacterium bovis bacille Calmette‐Guérin (BCG). We showed that Rab10 association with phagosomes is transient and confocal microscopy revealed detectible levels of Rab10 on phagosomal membranes at very early time‐points, occurring even before Rab5 acquisition. Rab10 recruitment had strong functional consequence, as the knockdown of endogenous Rab10 by RNA interference or overexpression of Rab10 dominant‐negative mutant delayed maturation of phagosomes of IgG‐opsonized latex beads or heat killed‐mycobacteria. These results can be explained, at least in part, by the involvement of Rab10 in recycling of some phagosomal components. More importantly, overexpression of the constitutively active mutant of Rab10 partially rescued live‐Mycobacterium‐containing phagosomes maturation. Indeed, we found that the membrane harbouring Mycobacterium acquired early endosome antigen 1 (EEA‐1), a marker excluded from phagosomes in control cells. Altogether these results indicate that Rab10, acting upstream of Rab5, plays a prominent role in phagolysosome formation and can modulate Mycobacterium‐containing phagosomes maturation.     

Comparative Genome Mapping of the Ataxia–Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Chromosomal locations of theAtm(ataxia–telangiectasia (AT)-mutated) andAcat1(mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4–qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice.Atm, Acat1,andNpat,which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among theAtm, Npat, Acat1,andD9Mit6loci, and these loci were mapped 2.0 cM distal toD9Mit99and 1.3 cM proximal toD9Mit102.Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion betweenEts1andAtm–Npat–Acat1and that the inversion of MMU9 originated from the chromosomal breakage at the boundary betweenGria4andAtm–Npat–Acat1on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with theRckgene.