scholarly journals Spatial segregation of the regulated and constitutive secretory pathways.

1989 ◽  
Vol 109 (1) ◽  
pp. 51-60 ◽  
Author(s):  
R J Rivas ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Cell Body ◽  
Secretory Granules ◽  
Membrane Receptors ◽  
Surface Glycoprotein ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Permissive Temperature ◽  
Secretory Pathways

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.

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 Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
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.

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 Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis.

1992 ◽  
Vol 118 (5) ◽  
pp. 1041-1056 ◽  
Author(s):  
R Bowser ◽  
H Müller ◽  
B Govindan ◽  
P Novick
Keyword(s):  
Plasma Membrane ◽  
Stop Codon ◽  
Gel Filtration ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Acid Region ◽  
Rich Domain ◽  
Downstream Effector ◽  
Acid Region

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.

scholarly journals Platelet and Megakaryocyte Dense Granules Contain Glycoproteins Ib and IIb-IIIa

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 4047-4057 ◽  
Author(s):  
Tayebeh Youssefian ◽  
Jean-Marc Massé ◽  
Francine Rendu ◽  
Josette Guichard ◽  
Elisabeth M. Cramer
Keyword(s):  
Plasma Membrane ◽  
Dense Body ◽  
Secretory Granules ◽  
Dense Granule ◽  
Immunogold Labeling ◽  
Membrane Receptors ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Plasma Membrane Receptors

Abstract Platelets contain two main types of secretory organelles, the dense granules and the α-granules. P-selectin, a specific receptor for leukocytes that is present in the α-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the α-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti–Gp IIb-IIIa or anti–Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti–Gp IIb-IIIa or anti–Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additionnal evidence of similarities between the α-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of α-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.

scholarly journals Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion

2009 ◽  
Vol 20 (3) ◽  
pp. 973-982 ◽  
Author(s):  
Jennifer A. Songer ◽  
Mary Munson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Protein Stability ◽  
Protein Complex ◽  
Mutational Analysis ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Conserved Residues ◽  
Temperature Sensitive Mutants ◽  
Exocyst Complex

The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.

scholarly journals The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst

2006 ◽  
Vol 17 (6) ◽  
pp. 2757-2769 ◽  
Author(s):  
Martina Medkova ◽  
Y. Ellen France ◽  
Jeff Coleman ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
Small Region ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Nucleotide Exchange ◽  
Vesicle Tethering ◽  
Exchange Factor ◽  
Polarized Transport ◽  
Recycling Pathway

Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.

scholarly journals In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles

2003 ◽  
Vol 370 (2) ◽  
pp. 641-649 ◽  
Author(s):  
Lorena ARRASTUA ◽  
Eider SAN SEBASTIAN ◽  
Ana F. QUINCOCES ◽  
Claude ANTONY ◽  
Unai UGALDE
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Membrane Vesicles ◽  
Fusion Process ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Plasma Membrane Vesicles ◽  
Fusion Event ◽  
Cytoplasmic Side

The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study.

scholarly journals The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

1997 ◽  
Vol 8 (7) ◽  
pp. 1361-1375 ◽  
Author(s):  
V Moreau ◽  
J M Galan ◽  
G Devilliers ◽  
R Haguenauer-Tsapis ◽  
B Winsor
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Endocytic Pathway ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Related Protein ◽  
Pathway Inhibition

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

scholarly journals Myosin motor proteins are involved in the final stages of the secretory pathways

2011 ◽  
Vol 39 (5) ◽  
pp. 1115-1119 ◽  
Author(s):  
Lisa M. Bond ◽  
Hemma Brandstaetter ◽  
James R. Sellers ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Plasma Membrane ◽  
Recent Work ◽  
Secretory Pathway ◽  
Motor Proteins ◽  
Myosin Ii ◽  
Secretory Vesicles ◽  
Secretory Pathways ◽  
Unconventional Myosin ◽  
Myosin Motor ◽  
Muscle Myosin

In eukaryotes, the final steps in both the regulated and constitutive secretory pathways can be divided into four distinct stages: (i) the ‘approach’ of secretory vesicles/granules to the PM (plasma membrane), (ii) the ‘docking’ of these vesicles/granules at the membrane itself, (iii) the ‘priming’ of the secretory vesicles/granules for the fusion process, and, finally, (iv) the ‘fusion’ of vesicular/granular membranes with the PM to permit content release from the cell. Recent work indicates that non-muscle myosin II and the unconventional myosin motor proteins in classes 1c/1e, Va and VI are specifically involved in these final stages of secretion. In the present review, we examine the roles of these myosins in these stages of the secretory pathway and the implications of their roles for an enhanced understanding of secretion in general.

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