TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells

1999 ◽  
Vol 112 (2) ◽  
pp. 147-156 ◽  
Author(s):  
H. Field ◽  
B.R. Ali ◽  
T. Sherwin ◽  
K. Gull ◽  
S.L. Croft ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Phylogenetic Analyses ◽  
Small Gtpases ◽  
Snare Complex ◽  
Rab Proteins ◽  
Over Expression ◽  
Golgi Transport

The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.

scholarly journals Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

1998 ◽  
Vol 9 (10) ◽  
pp. 2819-2837 ◽  
Author(s):  
Sara Jones ◽  
Celeste J. Richardson ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Assay

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7and GYP1, nor is it influenced by mutations inSEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

scholarly journals RINT-1 Regulates the Localization and Entry of ZW10 to the Syntaxin 18 Complex

2006 ◽  
Vol 17 (6) ◽  
pp. 2780-2788 ◽  
Author(s):  
Kohei Arasaki ◽  
May Taniguchi ◽  
Katsuko Tani ◽  
Mitsuo Tagaya
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Protein Transport ◽  
Snare Complex ◽  
Checkpoint Control ◽  
Interacting Protein ◽  
Golgi Transport ◽  
Radiation Induced ◽  
And Function

RINT-1 was first identified as a Rad50-interacting protein that participates in radiation-induced G2/M checkpoint control. We have recently reported that RINT-1, together with the dynamitin-interacting protein ZW10 and others, is associated with syntaxin 18, an endoplasmic reticulum (ER)-localized SNARE involved in membrane trafficking between the ER and Golgi. To address the role of RINT-1 in membrane trafficking, we examined the effects of overexpression and knockdown of RINT-1 on Golgi morphology and protein transport from the ER. Overexpression of the N-terminal region of RINT-1, which is responsible for the interaction with ZW10, caused redistribution of ZW10. Concomitantly, ER-to-Golgi transport was blocked and the Golgi was dispersed. Knockdown of RINT-1 also disrupted membrane trafficking between the ER and Golgi. Notably, silencing of RINT-1 resulted in a reduction in the amount of ZW10 associated with syntaxin 18, concomitant with ZW10 redistribution. In contrast, no redistribution or release of RINT-1 from the syntaxin 18 complex was observed when ZW10 expression was reduced. These results taken together suggest that RINT-1 coordinates the localization and function of ZW10 by serving as a link between ZW10 and the SNARE complex comprising syntaxin 18.

Rab proteins of the endoplasmic reticulum: functions and interactors

2012 ◽  
Vol 40 (6) ◽  
pp. 1426-1432 ◽  
Author(s):  
Carolina Ortiz Sandoval ◽  
Thomas Simmen
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Retrograde Trafficking ◽  
Rab Family ◽  
Intermediate Compartment

Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER–Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.

scholarly journals α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

2010 ◽  
Vol 21 (11) ◽  
pp. 1850-1863 ◽  
Author(s):  
Nandhakumar Thayanidhi ◽  
Jared R. Helm ◽  
Deborah C. Nycz ◽  
Marvin Bentley ◽  
Yingjian Liang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Direct Action ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Protein Receptors

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

Microsome-Based Assay for Analysis of Endoplasmic Reticulum to Golgi Transport in Mammalian Cells

Cell Biology ◽  
2006 ◽  
pp. 209-214 ◽  
Author(s):  
H PLUTNER ◽  
C GURKAN ◽  
X WANG ◽  
P LAPOINTE ◽  
W BALCH
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Golgi Transport

scholarly journals A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex

2013 ◽  
Vol 24 (18) ◽  
pp. 2907-2917 ◽  
Author(s):  
Kohei Arasaki ◽  
Daichi Takagi ◽  
Akiko Furuno ◽  
Miwa Sohda ◽  
Yoshio Misumi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Initial Contact ◽  
Complex Assembly ◽  
Trans Golgi Network ◽  
Cog Complex ◽  
Protein Receptor ◽  
Golgi Network

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor–mediated initial contact followed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum–localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to–trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

scholarly journals RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1089 ◽  
Author(s):  
Maeve Long ◽  
Tilen Kranjc ◽  
Margaritha M. Mysior ◽  
Jeremy C. Simpson
Keyword(s):  
Rna Interference ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Family Members ◽  
Membrane Traffic ◽  
Small Gtpases ◽  
Endomembrane System ◽  
Rho Family

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

2005 ◽  
Vol 72 ◽  
pp. 1-13 ◽  
Author(s):  
Krysten J. Palmer ◽  
Peter Watson ◽  
David J. Stephens
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Motor Proteins ◽  
Microtubule Cytoskeleton ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Intracellular Compartments ◽  
Retrograde Traffic

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.

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