scholarly journals The AP-1 complex is essential for fungal growth via its role in secretory vesicle polar sorting, endosomal recycling and cytoskeleton organization

2018 ◽  
Author(s):  
Olga Martzoukou ◽  
George Diallinas ◽  
Sotiris Amillis
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Fungal Growth ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Protein Traffic ◽  
Cytoskeleton Organization ◽  
Endosomal Recycling ◽  
Specific Membrane

AbstractThe AP-1 complex is essential for membrane protein traffic via its role in the pinching-off and sorting of secretory vesicles from the trans-Golgi and/or endosomes. While its essentiality is undisputed in metazoa, its role in model simpler eukaryotes seems less clear. Here we dissect the role of AP-1 in the filamentous fungus Aspergillus nidulans and show that it is absolutely essential for growth due to its role in clathrin-dependent maintenance of polar traffic of specific membrane cargoes towards the apex of growing hyphae. We provide evidence that AP-1 is involved in both anterograde sorting of RabERab11-labeled secretory vesicles and RabA/BRab5- dependent endosome recycling. Additionally, AP-1 is shown to be critical for microtubule and septin organization, further rationalizing its essentiality in cells that face the challenge of cytoskeleton-dependent polarized cargo traffic. This work also opens a novel issue on how non-polar cargoes, such as transporters, are sorted to the eukaryotic plasma membrane.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

scholarly journals Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

2001 ◽  
Vol 155 (4) ◽  
pp. 581-592 ◽  
Author(s):  
Joan E. Adamo ◽  
John J. Moskow ◽  
Amy S. Gladfelter ◽  
Domenic Viterbo ◽  
Daniel J. Lew ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Vesicle Docking ◽  
Actin Organization ◽  
Late Step ◽  
Rho Family ◽  
Localization Data

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

scholarly journals Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

1997 ◽  
Vol 8 (8) ◽  
pp. 1481-1499 ◽  
Author(s):  
J Mulholland ◽  
A Wesp ◽  
H Riezman ◽  
D Botstein
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Biochemical Analysis ◽  
Small Gtpase ◽  
Secretory Vesicles ◽  
Cytoskeleton Organization ◽  
New Class ◽  
Actin Cytoskeleton Organization ◽  
Vectorial Transport

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

scholarly journals Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

1994 ◽  
Vol 299 (2) ◽  
pp. 473-479 ◽  
Author(s):  
H Sengeløv ◽  
F Boulay ◽  
L Kjeldsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Beta Band ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

Molecular mechanism of secretory vesicle docking

2010 ◽  
Vol 38 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Heidi de Wit
Keyword(s):  
Plasma Membrane ◽  
Cell Model ◽  
Secretory Vesicle ◽  
Model Systems ◽  
Secretory Vesicles ◽  
Filamentous Actin ◽  
Embryonic Mouse ◽  
Vesicle Docking ◽  
Bovine Chromaffin Cell ◽  
Stable Association

Docking, the stable association of secretory vesicles with the plasma membrane, is considered to be the necessary first step before vesicles gain fusion-competence, but it is unclear how vesicles dock. In adrenal medullary chromaffin cells, access of secretory vesicles to docking sites is controlled by dense F-actin (filamentous actin) beneath the plasma membrane. Recently, we found that, in the absence of Munc18-1, the number of docked vesicles and the thickness of cortical F-actin are affected. In the present paper, I discuss the possible mechanism by which Munc18-1 modulates cortical F-actin and how it orchestrates the docking machinery via an interaction with syntaxin-1. Finally, a comparison of Munc18's role in embryonic mouse and adult bovine chromaffin cell model systems will be made to clarify observed differences in cortical F-actin as well as docking phenotypes.

scholarly journals Relative Contribution of PIN-containing Secretory Vesicles and Plasma Membrane PINs to the Directed Auxin Transport: Theoretical Estimation

2018 ◽  
Author(s):  
Sander Hille ◽  
Maria Akhmanova ◽  
Matouš Glanc ◽  
Alexander Johnson ◽  
Jiří Friml
Keyword(s):  
Plasma Membrane ◽  
Auxin Transport ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Transport Mechanisms ◽  
In Planta ◽  
Major Mechanism ◽  
Relative Contribution ◽  
Secretory Transport ◽  
Auxin Flux

Intercellular transport of auxin is driven by PIN-formed (PIN) proteins. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping it into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D-SIM microscopy was used to determine PIN density on the PM. Combing this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000x greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is intriguing and theoretically possible model, it unlikely to be a major mechanism of auxin transport in planta.

scholarly journals Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

2021 ◽  
Vol 22 (21) ◽  
pp. 11727
Author(s):  
Maria J. Sarmento ◽  
Luís Borges-Araújo ◽  
Sandra N. Pinto ◽  
Nuno Bernardes ◽  
Joana C. Ricardo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Functions ◽  
Cytoskeleton Organization

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

scholarly journals Effects of ATP9A on Extracellular Vesicle Release and Exosomal Lipid Composition

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Xiao Xu ◽  
Limei Xu ◽  
Peng Zhang ◽  
Kan Ouyang ◽  
Yin Xiao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Lipid Composition ◽  
Extracellular Vesicle ◽  
Biological Processes ◽  
Vesicle Release ◽  
Endosomal Recycling ◽  
Intracellular Compartments ◽  
Intercellular Communications

Numerous biological processes are regulated by the intercellular communications arising from extracellular vesicles (EVs) released from cells. However, the mechanisms that regulate the quantity of EV discharged have yet to be understood. While it is known that ATP9A, a P4-ATPase, is involved in endosomal recycling, it is not clear whether it also contributes to the release of EVs and the makeup of exosomal lipids. This study is aimed at exploring the role of human ATP9A in the process of EV release and, further, to analyze the profiles of EV lipids regulated by ATP9A. Our results demonstrate that ATP9A is located in both the intracellular compartments and the plasma membrane. The percentage of ceramides and sphingosine was found to be significantly greater in the control cells than in the ATP9A overexpression and ATP9A knockout groups. However, EV release was greater in ATP9A knockout cells, indicating that ATP9A inhibits the release of EVs. This study revealed the effects of ATP9A on the release of EVs and the lipid composition of exosomes.

scholarly journals PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy in Pichia pastoris

2005 ◽  
Vol 16 (10) ◽  
pp. 4941-4953 ◽  
Author(s):  
Tina Chang ◽  
Laura A. Schroder ◽  
J. Michael Thomson ◽  
Amy S. Klocman ◽  
Amber J. Tomasini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pichia Pastoris ◽  
Membrane Protein ◽  
Peripheral Compartment ◽  
Membrane Apparatus ◽  
Multiple Structures ◽  
Vacuolar Membranes ◽  
Specific Membrane

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.

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