Morphological and histochemical characterization of the secretory epithelium in the canine lacrimal gland

In the present study, the expression of secretory components and vesicular transport proteins in the canine lacrimal gland was examined and morphometric analysis was performed. The secretory epithelium consists of two types of secretory cells with different morphological features. The secretory cells constituting acinar units (type A cells) exhibited higher levels of glycoconjugates, including β-GlcNAc, than the other cell type constituting tubular units (type T cells). Immunoblot analysis revealed that antimicrobial proteins, such as lysozyme, lactoferrin and lactoperoxidase, Rab proteins (Rab3d, Rab27a and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins (VAMP2, VAMP4, VAMP8, syntaxin-1, syntaxin-4 and syntaxin-6), were expressed at various levels. We immunohistochemically demonstrated that the expression patterns of lysozyme, lactoferrin, Rab27a, Rab27b, VAMP4, VAMP8 and syntaxin-6 differed depending on the secretory cell type. Additionally, in type T cells, VAMP4 was confined to a subpopulation of secretory granules, while VAMP8 was detected in almost all of them. The present study displayed the morphological and histochemical characteristics of the secretory epithelium in the canine lacrimal gland. These findings will help elucidate the species-specific properties of this gland.Key words: dog, lacrimal gland, glycoconjugate, Rab protein, SNARE protein, electron microscopy     

Regulation of the cell surface expression and function of angiotensin II type 1 receptor by Rab1-mediated endoplasmic reticulum-to-Golgi transport in cardiac myocytes

Rab1 GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ER-to-Golgi transport as a regulatory site for control of cardiomyocyte growth.     

ICA69 is a novel Rab2 effector regulating ER-Golgi trafficking in insulinoma cells

Islet cell autoantigen of 69kDa (ICA69) is a small GTPase-binding protein of unknown function. ICA69 is enriched in the Golgi complex and its N-terminal half contains a BAR domain, a module that can bind/bend membranes and interacts with phospholipids. Here we show that in insulinoma INS-1 cells ICA69 binds to the small GTPase Rab2, which regulates the transport of COPI vesicles between the endoplasmic reticulum and the Golgi complex. Rab2 binds to ICA69 in a GTP-dependent fashion and recruits it to membranes. Over-expression of either Rab2 or ICA69 in INS-1 cells results in a phenotype characterized by: (i) impaired anterograde transport of the secretory granule protein precursors pro-ICA512 and chromogranin A; (ii) reduction of stimulated insulin secretion. Taken together, these data identify ICA69 as a novel Rab2 effector and point to its role in regulating the early transport of insulin secretory granule proteins.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Rab1 GTPase promotes expression of β-adrenergic receptors in rat pulmonary microvascular endothelial cells

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (β-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous β-ARs in RPMVECs in the presence of lipopolysaccharide (LPS).We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of β-ARs on the cell surface, whereas the dominant-negative mutant Rab1N124I significantly attenuated β-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced β-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by over-expression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged β₂-AR in the ER. Consistent with their effects on β-ARs export, Rab1WT and Rab1N124I differentially modified the β-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyper-permeability of RPMVECs by increasing the expression of β₂-AR on the cell surface. These data reveal that β-ARs function in RPMVECs could be modulated by manipulating β-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs.     

mTrs130 Is a Component of a Mammalian TRAPPII Complex,a Rab1 GEF That Binds to COPI-coated Vesicles

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.     

Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

Background

The 60⁺ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a’, Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a’ has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization.

Methods/Results

Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel.

Conclusion

We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a’ and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our data indicate that Rab VI subfamily members, although related by homology and structure, share limited functional conservation.     

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.     

Rab26 Modulates the Cell Surface Transport of α2-Adrenergic Receptors from the Golgi

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α₂-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α_2A-AR and α_2B-AR, as well as ERK1/2 activation by α_2B-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α_2B-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α₂-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.     

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.     

PERK inhibition attenuates the abnormalities of the secretory pathway and the increased apoptotic rate induced by SIL1 knockdown in HeLa cells

Loss-of-function mutations in the SIL1 gene are linked to Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of infancy characterized by cerebellar and skeletal muscle degeneration. SIL1 is a ubiquitous adenine nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone BiP. The complexity of mechanisms by which loss of SIL1 causes MSS is not yet fully understood. We used HeLa cells to test the hypothesis that impaired protein folding in the ER due to loss of SIL1 could affect secretory trafficking, impairing the transport of cargoes essential for the function of MSS vulnerable cells. Immunofluorescence and ultrastructural analysis of SIL1-knocked-down cells detected ER chaperone aggregation, enlargement of the Golgi complex, increased autophagic vacuoles, and mitochondrial swelling. SIL1-interefered cells also had delayed ER-to-plasma membrane transport with retention of Na⁺/K⁺-ATPase and procollagen-I in the ER and Golgi, and increased apoptosis. The PERK pathway of the unfolded protein response was activated in SIL1-interfered cells, and the PERK inhibitor GSK2606414 attenuated the morphological and functional alterations of the secretory pathway, and significantly reduced cell death. These results indicate that loss of SIL1 is associated with alterations of secretory transport, and suggest that inhibiting PERK signalling may alleviate the cellular pathology of SIL1-related MSS.     

Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein,in mouse testes

Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.     

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.