Rab5 regulates macropinocytosis by recruiting the inositol 5-phosphatases OCRL and Inpp5b that hydrolyse PtdIns(4,5)P2

ABSTRACT Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

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Rab5 regulates macropinosome closure through recruitment of the inositol 5-phosphatases OCRL/Inpp5b and the hydrolysis of PtdIns(4,5)P2

10.1101/2020.06.08.139436 ◽

2020 ◽

Author(s):

Michelle E. Maxson ◽

Helen Sarantis ◽

Allen Volchuk ◽

John H. Brumell ◽

Sergio Grinstein

Keyword(s):

Plasma Membrane ◽

Mode Of Action ◽

Dominant Negative ◽

Circular Membrane ◽

Fusion Event ◽

Phosphatidylinositol 4 ◽

Hydrolysis Of

AbstractRab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete macropinosome scission from the plasmalemma.

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Insulin and Hypertonicity Recruit GLUT4 to the Plasma Membrane of Muscle Cells by Using N-Ethylmaleimide-sensitive Factor-dependent SNARE Mechanisms but Different v-SNAREs: Role of TI-VAMP

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0266 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5565-5573 ◽

Cited By ~ 43

Author(s):

Varinder K. Randhawa ◽

Farah S.L. Thong ◽

Dawn Y. Lim ◽

Dailin Li ◽

Rami R. Garg ◽

...

Keyword(s):

Plasma Membrane ◽

Muscle Cells ◽

Dominant Negative ◽

Attachment Protein ◽

Insulin Effect ◽

Tetanus Neurotoxin ◽

Protein Receptor ◽

Sensitive Factor ◽

Interfering Rna

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

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Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1368 ◽

2015 ◽

Vol 26 (3) ◽

pp. 530-536 ◽

Cited By ~ 15

Author(s):

Jessica B. A. Sadler ◽

Nia J. Bryant ◽

Gwyn W. Gould

Keyword(s):

Plasma Membrane ◽

Snare Complex ◽

Cell Type ◽

Attachment Protein ◽

Systematic Analysis ◽

Protein Receptor ◽

Sensitive Factor ◽

Factor Attachment Protein Receptor

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

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News about non-secretory exocytosis: mechanisms, properties, and functions

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjy084 ◽

2019 ◽

Vol 11 (9) ◽

pp. 736-746 ◽

Cited By ~ 1

Author(s):

Rosalba D’Alessandro ◽

Jacopo Meldolesi

Keyword(s):

Plasma Membrane ◽

Extracellular Space ◽

The Other ◽

Membrane Repair ◽

Attachment Protein ◽

Receptor Complexes ◽

Protein Receptor ◽

Sensitive Factor ◽

Golgi Network ◽

Factor Attachment Protein Receptor

AbstractThe fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes. Other exocytic vesicles, however, do not contain cargoes, and thus, their fusion is not followed by secretion. Therefore, two distinct processes of exocytosis exist, one secretory and the other non-secretory. The present review deals with the knowledge of non-secretory exocytosis developed during recent years. Among such developments are the dual generation of the exocytic vesicles, initially released either from the trans-Golgi network or by endocytosis; their traffic with activation of receptors, channels, pumps, and transporters; the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions; the growth of axons and the membrane repair. Examples of potential relevance of these processes for pathology and medicine are also reported. The developments presented here offer interesting chances for future progress in the field.

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A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0010 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3379-3389 ◽

Cited By ~ 9

Author(s):

Elena Fdez ◽

Thomas A. Jowitt ◽

Ming-Chuan Wang ◽

Manisha Rajebhosale ◽

Keith Foster ◽

...

Keyword(s):

Dominant Negative ◽

Neuroendocrine Cells ◽

Snare Complex ◽

Membrane Interface ◽

Attachment Protein ◽

Protein Receptor ◽

Sensitive Factor ◽

The Stability ◽

Factor Attachment Protein Receptor

The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicle-associated SNARE synaptobrevin 2 that lie close to the cytosol–membrane interface within the full-length protein. Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion.

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