BIG3 and BIG5 Redundantly Mediate Vesicle Trafficking in Arabidopsis

Vesicle trafficking plays an important role in delivering a diverse range of cargoes between different membranous systems in eukaryotes. It is well documented that the brefeldin A (BFA)-inhibited guanine nucleotide exchange factor (GEF), named BIG, regulates vesicle budding at the trans-Golgi network (TGN) and recycling endosomes through activating the ADP-ribosylation factor (ARFs). Among the five BIGs in Arabidopsis, BIG5 is characterized to mediate ARF-dependent trafficking at the plasma membrane or endosomes while the members from BIG1 to BIG4 (BIG1-BIG4) at the TGN in the secretory pathway. However, evidence is increasing to suggest that BIG5 can function redundantly with BIG1-BIG4 to regulate vesicular trafficking in response to various intra- and extra-cellular stimuli. In this study, our genetic analysis showed that BIG5 played an overlapping role at least with BIG3 in cell proliferation. To elucidate molecular mechanisms underlying the BIG5- and BIG3-regulated biological processes, we examined the effect of BIGs on expression patterns of the two transmembrane proteins, PINFORMED 2 (PIN2) epically localized in root epidermal cells and the regulator of G protein signaling 1 (RGS1) localized in the plasma membrane. Our data showed that the PIN2 polar distribution was slightly reduced in big3 big5 in the absence of BFA, and it was significantly reduced by the treatment of 0.1 µM BFA in big3 big5. Further analysis revealed that BFA bodies derived from the plasma membrane were only observed in wild type (WT), big3 and big5 cells, but not in the big3 big5 cells. These results indicate that BIG5 and BIG3 are functionally redundant in the endosome recycling pathway from the plasma membrane to TGN. On the other hand, the single BIG3 or BIG5 mutation had no effect on the plasma membrane expression of RGS1, whereas the double mutations in BIG3 and BIG5 led to a significant amount of RGS1 retained in the vesicle, indicating that BIG3 and BIG5 act redundantly in mediating protein trafficking. Furthermore, transmission electron microscopy assays showed that Golgi ultrastructure in big3 big5 cells was abnormal and similar to that in BFA-treated WT cells. Taken together, our data provide several new lines of evidence supporting that BIGs play a redundant role in vesicular trafficking and probably also in maintaining the Golgi structural integrity in Arabidopsis.

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Retrograde Transport of Golgi-localized Proteins to the ER

The Journal of Cell Biology ◽

10.1083/jcb.140.1.1 ◽

1998 ◽

Vol 140 (1) ◽

pp. 1-15 ◽

Cited By ~ 153

Author(s):

Nelson B. Cole ◽

Jan Ellenberg ◽

Jia Song ◽

Diane DiEuliis ◽

Jennifer Lippincott-Schwartz

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Fusion Proteins ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Viral Glycoprotein ◽

Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

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Understanding the Matrix: The Role of Extracellular DNA in Oral Biofilms

Frontiers in Oral Health ◽

10.3389/froh.2021.640129 ◽

2021 ◽

Vol 2 ◽

Author(s):

Hannah J. Serrage ◽

Mark A. Jepson ◽

Nadia Rostami ◽

Nicholas S. Jakubovics ◽

Angela H. Nobbs

Keyword(s):

Dental Plaque ◽

Molecular Mechanisms ◽

Structural Integrity ◽

Extracellular Dna ◽

Oral Disease ◽

Diverse Range ◽

Oral Biofilms ◽

The Matrix ◽

Plaque Biofilm

Dental plaque is the key etiological agent in caries formation and the development of the prevalent chronic oral inflammatory disease, periodontitis. The dental plaque biofilm comprises a diverse range of microbial species encased within a rich extracellular matrix, of which extracellular DNA (eDNA) has been identified as an important component. The molecular mechanisms of eDNA release and the structure of eDNA have yet to be fully characterized. Nonetheless, key functions that have been proposed for eDNA include maintaining biofilm structural integrity, initiating adhesion to dental surfaces, acting as a nutrient source, and facilitating horizontal gene transfer. Thus, eDNA is a potential therapeutic target for the management of oral disease–associated biofilm. This review aims to summarize advances in the understanding of the mechanisms of eDNA release from oral microorganisms and in the methods of eDNA detection and quantification within oral biofilms.

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An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101287118 ◽

2021 ◽

Vol 118 (35) ◽

pp. e2101287118

Author(s):

Yan Huang ◽

Haidi Yin ◽

Baiying Li ◽

Qian Wu ◽

Yang Liu ◽

...

Keyword(s):

Secretory Pathway ◽

Molecular Mechanisms ◽

Protein Transport ◽

Vesicular Trafficking ◽

Vesicle Formation ◽

Dependent Manner ◽

Vitro Assay ◽

Cargo Proteins ◽

Er Exit Sites

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

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Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1

AJP Renal Physiology ◽

10.1152/ajprenal.00226.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. F335-F343 ◽

Cited By ~ 11

Author(s):

J. Reinhardt ◽

M. Kosch ◽

M. Lerner ◽

H. Bertram ◽

D. Lemke ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Phorbol Esters ◽

Ion Pump ◽

Membrane Expression ◽

Kinase C ◽

Plasma Membrane Expression ◽

Functional Inactivation

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.

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Regulation of GPR54 Signaling by GRK2 and β-Arrestin

Molecular Endocrinology ◽

10.1210/me.2009-0013 ◽

2009 ◽

Vol 23 (12) ◽

pp. 2060-2074 ◽

Cited By ~ 60

Author(s):

Macarena Pampillo ◽

Natasha Camuso ◽

Jay E. Taylor ◽

Jacob M. Szereszewski ◽

Maryse R. Ahow ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Line ◽

Molecular Mechanisms ◽

Control Cell ◽

Cytoplasmic Tail ◽

Intracellular Loop ◽

Membrane Expression ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Abstract Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a Gq/11-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca2+ mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and β-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less β-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and β- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that β-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

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RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic

International Journal of Molecular Sciences ◽

10.3390/ijms22137087 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7087

Author(s):

Amel Ben Saad ◽

Virginie Vauthier ◽

Martine Lapalus ◽

Elodie Mareux ◽

Evangéline Bennana ◽

...

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Significant Proportion ◽

Deep Understanding ◽

Small Gtpase ◽

Membrane Expression ◽

Intracellular Traffic ◽

And Function ◽

Cholestatic Diseases ◽

Phosphatidylcholine Secretion

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

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Study of Protein Targeting and Mislocalization in Rod Photoreceptors

Proceedings of IMPRS ◽

10.18060/25714 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Asael Nunez ◽

Shimpei Takita ◽

Sanae Imanishi ◽

Yoshikazu Imanishi

Keyword(s):

Plasma Membrane ◽

Mouse Model ◽

Retinitis Pigmentosa ◽

Retinal Degeneration ◽

Molecular Mechanisms ◽

Major Protein ◽

Membrane Expression ◽

Rich Region ◽

Rod Photoreceptors ◽

Proline Rich Region

The photoreceptor outer segment (OS) is a highly specialized organelle for light absorption. Precise localization of OS resident proteins is important for photoreceptor function. Molecular mechanisms underlying OS targeting of proteins and their mislocalization, which frequently causes inherited retinal degeneration, have been intensely investigated. Rhodopsin, a major protein of the rod OS, is often mislocalized to the inner segment (IS) plasma membrane of rod photoreceptors in retinal degeneration patients. In the Xenopus laevis model of retinitis pigmentosa, we previously found that Na+/K+-ATPase (NKA), a major IS protein, was downregulated. The Imanishi lab recently created a novel retinitis pigmentosa mouse model carrying the Q344ter rhodopsin gene mutation, which causes rhodopsin mislocalization to the rod IS plasma membrane. In this summer program, we examined whether this mouse model also displays reduced NKA expression in the rod IS’s by immunohistochemistry at postnatal day 30. Although NKA was properly localized to the IS plasma membrane, expression of NKA was reduced in mutant photoreceptors compared to wildtype cells. In the rod OS, activation of rhodopsin eventually leads to the closure of the cyclic nucleotide gated (CNG) channel, which consists of a and b subunits. This channel localizes to the OS plasma membrane, and the N-terminal proline-rich region (R) of the b subunit (CNGb1) may be important for its interaction with peripherin (PRPH2), another OS resident protein. Currently, it is not well understood whether this interaction is necessary for the proper localization of CNGb1 to the OS plasma membrane. Using Xenopus as a model, we studied the role of the N-terminal proline-rich region in properly localizing CNGb1 to the OS plasma membrane by generating transgenic CNGb1(DR) tadpoles that expressed CNGb1(DR) in rods under the control of a rhodopsin promoter. We found that CNGb1(DR) properly localized to the OS plasma membrane.

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Protein Sorting in Plasmodium Falciparum

Life ◽

10.3390/life11090937 ◽

2021 ◽

Vol 11 (9) ◽

pp. 937

Author(s):

D.C. Ghislaine Mayer

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Protein Sorting ◽

Vesicular Trafficking ◽

Unicellular Eukaryote ◽

Dense Granules ◽

Host Cell Proteins ◽

Endosymbiotic Organelle

Plasmodium falciparum is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. P. falciparum, like its relative T. gondii, uses the endolysosomal system to produce the secretory organelles and to ingest host cell proteins. The parasite also has an apicoplast, a secondary endosymbiotic organelle, which depends on vesicular trafficking for appropriate incorporation of nuclear-encoded proteins into the apicoplast. Recently, the central molecules responsible for sorting and trafficking in P. falciparum and T. gondii have been characterized. From these studies, it is now evident that P. falciparum has repurposed the molecules of the endosomal system to the secretory pathway. Additionally, the sorting and vesicular trafficking mechanism seem to be conserved among apicomplexans. This review described the most recent findings on the molecular mechanisms of protein sorting and vesicular trafficking in P. falciparum and revealed that P. falciparum has an amazing secretory machinery that has been cleverly modified to its intracellular lifestyle.

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Phosphoinositides and membrane traffic at the trans-Golgi network.

Biochemical Society Symposium ◽

10.1042/bss0720031 ◽

2005 ◽

Vol 72 ◽

pp. 31-38 ◽

Cited By ~ 11

Author(s):

Rawshan R. Choudhury ◽

Noora Hyvola ◽

Martin Lowe

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Membrane Traffic ◽

Trans Golgi Network ◽

Cargo Proteins ◽

Golgi Network

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.

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WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

AJP Renal Physiology ◽

10.1152/ajprenal.90229.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. F1472-F1484 ◽

Cited By ~ 26

Author(s):

Wei Zhang ◽

Tao Na ◽

Ji-Bin Peng

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Kinase Domain ◽

Xenopus Laevis Oocytes ◽

Membrane Expression ◽

Transport Pathway ◽

Microtubule Inhibitor ◽

Plasma Membrane Expression ◽

Cation Chloride Cotransporters ◽

Dependent Mechanism

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12A family of cation-chloride cotransporters and the renal outer medullary K+ channel ROMK and Cl− channel SLC26A9. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 and TRPV5 were coexpressed in Xenopus laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 82.7 ± 7.1% increase in TRPV5-mediated Ca2+ uptake was observed when WNK3 was coexpressed. A similar increase in TRPV5-mediated Na+ current was observed with the voltage-clamp technique. WNK3 also enhanced Ca2+ influx and Na+ current mediated by TRPV6, which is the closest homolog of TRPV5 that mediates active intestinal Ca2+ absorption. The kinase domain of WNK3 alone was sufficient to increase TRPV5-mediated Ca2+ transport, and the positive regulatory effect was abolished by the kinase-inactive D294A mutation in WNK3, indicating a kinase-dependent mechanism. The complexly glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3. The exocytosis of TRPV5 was increased by WNK3, and the effect of WNK3 on TRPV5 was abolished by the microtubule inhibitor colchicine. The increased plasma membrane expression of TRPV5 was likely due to the enhanced delivery of mature TRPV5 to the plasma membrane from its intracellular pool via the secretory pathway. These results indicate that WNK3 is a positive regulator of the transcellular Ca2+ transport pathway.

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