Identification and characterization of Noc2 as a potential Rab3B effector protein in epithelial cells

The Rab3 family small G proteins (Rab3A-D) are involved in the regulated secretory pathway of brain and secretory tissues. Among Rab3-interacting proteins, Rabphilin-3, Rim, and Noc2, all of which contain a conserved Rab3-binding domain (RBD3), are generally recognized Rab3 effector proteins in neurons and secretory cells. Although Rab3B was also detected in epithelial cells, its function remained unknown. We isolated cDNA sequences from human epithelial Caco2-cell mRNA by degenerate RT-PCR based on the conserved amino acid sequence of RBD3. Multiple cDNA clones were identified as encoding Noc2. Northern blot analysis revealed that Noc2 mRNA was expressed not only in secretory tissues but also in epithelial tissues and cell lines. A pull-down assay demonstrated that Noc2 bound to Rab3B in a GTP-dependent manner. When Noc2 was co-expressed with the GTP-bound form of Rab3B, it was recruited from the cytosol to perinuclear membranes. Furthermore, overexpression of Noc2 inhibited the cell-surface transport of basolateral vesicular stomatitis virus glycoprotein. These results suggest that Noc2 functions as a potential Rab3B effector protein in epithelial cells.     

A general role for Rab27a in secretory cells

Vesicular transport is a complex multistep process regulated by distinct Rab GTPases. Here, we show for the first time that an EGFP-Rab fusion protein is fully functional in a mammalian organism. We constructed a PAC-based transgenic mouse, which expresses EGFP-Rab27a under the control of endogenous Rab27a promoter. The EGFP-Rab27a transgene was fully functional and rescued the two major defects of the ashen Rab27a knockout mouse. We achieved cell-specific expression of EGFP-Rab27a, which faithfully followed the pattern of expression of endogenous Rab27a. We found that Rab27a is expressed in an exceptionally broad range of specialized secretory cells, including exocrine (particularly in mucin- and zymogen-secreting cells), endocrine, ovarian, and hematopoietic cells, most of which undergo regulated exocytosis. We suggest that Rab27a acts in concert with Rab3 proteins in most regulated secretory events. The present strategy represents one way in which the complex pattern of expression and function of proteins involved in specialized cell types may be unraveled.     

Loss of Rab27 function results in abnormal lung epithelium structure in mice

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.     

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.     

Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251bp with an open reading frame (ORF) of 636bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.     

The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis

The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.     

Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.     

Localization and functional analysis of the insect‐specific RabX4 in the brain of Bombyx mori

Rab proteins are small monomeric GTPases/GTP‐binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect‐specific Rab protein that has no close homolog in vertebrates. There is little information about insect‐specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4‐like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.     

Emerging roles for Rab family GTPases in human cancer

Members of the Ras-associated binding (Rab) family of small GTPases function as molecular switches regulating vesicular transport in eukaryotes cells. Their pathophysiological roles in human malignancies are less well-known compared to members of Ras and Rho families. Several members of the Rab family have, however, been shown to be aberrantly expressed in various cancer tissues. Recent findings have also revealed, in particular, Rab25 as a determinant of tumor progression and aggressiveness of epithelial cancers. Rab25 associates with α5β1 integrin, and enhances tumor cell invasion by directing the localization of integrin-containing vesicles to the leading edge of matrix invading pseudopodia. We summarized here recent findings on Rab25 and other Rabs implicated to be involved in a variety of human cancers, and discussed plausible mechanisms of how dysregulation of Rab expression could be tumorigenic or tumor suppressive.     

Rab27 Effectors,Pleiotropic Regulators in Secretory Pathways

Rab27, a member of the small GTPase Rab family, is widely conserved in metazoan, and two Rab27 isoforms, Rab27A and Rab27B, are present in vertebrates. Rab27A was the first Rab protein whose dysfunction was found to cause a human hereditary disease, type 2 Griscelli syndrome, which is characterized by silvery hair and immunodeficiency. The discovery in the 21st century of three distinct types of mammalian Rab27A effectors [synaptotagmin‐like protein (Slp), Slp homologue lacking C2 domains (Slac2), and Munc13‐4] that specifically bind active Rab27A has greatly accelerated our understanding not only of the molecular mechanisms of Rab27A‐mediated membrane traffic (e.g. melanosome transport and regulated secretion) but of the symptoms of Griscelli syndrome patients at the molecular level. Because Rab27B is widely expressed in various tissues together with Rab27A and has been found to have the ability to bind all of the Rab27A effectors that have been tested, Rab27A and Rab27B were initially thought to function redundantly by sharing common Rab27 effectors. However, recent evidence has indicated that by interacting with different Rab27 effectors Rab27A and Rab27B play different roles in special types of secretion (e.g. exosome secretion and mast cell secretion) even within the same cell type. In this review article, I describe the current state of our understanding of the functions of Rab27 effectors in secretory pathways .     

Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.     

The Rab27 effector Rabphilin, unlike Granuphilin and Noc2, rapidly exchanges between secretory granules and cytosol in PC12 cells

Rab proteins are GTPases that transit between GTP- and GDP-bound states. In the GTP-bound form they can recruit specific effector to membrane domains. It is possible that the exchange of Rab effectors between membranes and cytosol would be determined by the exchange of the particular Rab partner. We have compared the cycling of three Rab3/27 effectors, Granuphilin, Noc2, and Rabphilin, in PC12 cells using fluorescence recovery after photobleaching of EGFP-tagged proteins. All three effectors become localised to secretory granules. Granuphilin and Noc2 showed little or no exchange between secretory granules and cytosol whereas Rabphilin showed rapid and complete exchange. Both Noc2 and Rabphilin were found to be recruited to granules by Rab27 but the data suggest that Rabphilin did not form stable complexes with Rab27 on secretory granules and so Rab effector cycling between membranes and cytosol can be independent of that of the Rab protein.     

Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.     

Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin

Rab GTPases associated with insulin‐containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β‐cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase‐activating protein overexpression in β‐cells from wild‐type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP‐bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release‐ready SGs in β‐cells, they also direct unique kinetic and functional properties of the exocytotic pathway.      

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.     

Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism

Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for embryonic nervous system development in <Emphasis Type="Italic">Drosophila</Emphasis>

In eukaryotes, membrane trafficking is regulated by the small monomeric GTPases of Rab protein family. Rab11, an evolutionary conserved, ubiquitously expressed subfamily of the Rab GTPases, has been implicated in the regulation of vesicular trafficking through the recycling of endosomes. To dissect out the role of this protein during embryonic nervous system development, we have studied the expression pattern of Rab11 in the ventral nerve cord during neuronal differentiation in the Drosophila embryo. When the dominant-negative or constitutively-active mutant DRab11 proteins are expressed in neurons, or when homozygous mutant Rab11 embryos are analyzed, defects are found in the developing central nervous system, along with disorganization and misrouting of embryonic axons. Our results provide the first in vivo evidence that Rab11 is involved in the development of the nervous system during Drosophila embryogenesis. This work was supported by the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.     

Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.