scholarly journals Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

2010 ◽  
Vol 188 (4) ◽  
pp. 537-546 ◽  
Author(s):  
Ravi Manjithaya ◽  
Christophe Anjard ◽  
William F. Loomis ◽  
Suresh Subramani
Keyword(s):  
Pichia Pastoris ◽  
Fatty Acyl ◽  
Autophagosome Formation ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Evolutionarily Conserved ◽  
Unconventional Secretion ◽  
Acyl Coenzyme A ◽  
And Function ◽  
Protein Attachment

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)–binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2–like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

Regulation of SNAP-25 trafficking and function by palmitoylation

2010 ◽  
Vol 38 (1) ◽  
pp. 163-166 ◽  
Author(s):  
Jennifer Greaves ◽  
Gerald R. Prescott ◽  
Oforiwa A. Gorleku ◽  
Luke H. Chamberlain
Keyword(s):  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Receptor Protein ◽  
Attachment Protein ◽  
Rich Region ◽  
Protein Receptor ◽  
And Function ◽  
Protein Attachment

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25 is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review, we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular trafficking and exocytotic function of this essential protein.

scholarly journals Comparative analysis of tandem C2 domains from the mammalian synaptotagmin family

2004 ◽  
Vol 378 (2) ◽  
pp. 681-686 ◽  
Author(s):  
Colin RICKMAN ◽  
Molly CRAXTON ◽  
Shona OSBORNE ◽  
Bazbek DAVLETOV
Keyword(s):  
Membrane Trafficking ◽  
Sequence Similarity ◽  
Attachment Protein ◽  
C2 Domains ◽  
Phospholipid Binding ◽  
Calcium Dependent ◽  
Functional Studies ◽  
Protein Receptor ◽  
High Degree ◽  
Protein Attachment

Intracellular membrane traffic is governed by a conserved set of proteins, including Syts (synaptotagmins). The mammalian Syt family includes 15 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium-dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison, together with functional studies of rat Syt C2ABs, to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Nearly half of the Syt family does not exhibit calcium/phospholipid binding in comparison to Syt I, the major brain isoform. Syts do, however, possess a more conserved function, namely calcium-independent binding to target SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) heterodimers. All tested isoforms, except Syt XII and Syt XIII, bound the target SNARE heterodimer comprising syntaxin 1 and SNAP-25 (25 kDa synaptosome-associated protein). Our present study suggests that many Syt isoforms can function in membrane trafficking to interact with the target SNARE heterodimer on the pathway to calcium-triggered membrane fusion.

scholarly journals The yeast Batten disease orthologue Btn1 controls endosome–Golgi retrograde transport via SNARE assembly

2011 ◽  
Vol 195 (2) ◽  
pp. 203-215 ◽  
Author(s):  
Rachel Kama ◽  
Vydehi Kanneganti ◽  
Christian Ungermann ◽  
Jeffrey E. Gerst
Keyword(s):  
Disease Gene ◽  
Transmembrane Domain ◽  
Batten Disease ◽  
Retrograde Transport ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Target Membrane ◽  
Opposing Effects ◽  
Protein Attachment

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE–Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

The molecular mechanisms of the mammalian exocyst complex in exocytosis

2006 ◽  
Vol 34 (5) ◽  
pp. 687-690 ◽  
Author(s):  
S. Wang ◽  
S.C. Hsu
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Secretory Vesicles ◽  
Attachment Protein ◽  
Trafficking Pathway ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Associated Proteins ◽  
Protein Attachment

Exocytosis is a highly ordered vesicle trafficking pathway that targets proteins to the plasma membrane for membrane addition or secretion. Research over the years has discovered many proteins that participate at various stages in the mammalian exocytotic pathway. At the early stage of exocytosis, co-atomer proteins and their respective adaptors and GTPases have been shown to play a role in the sorting and incorporation of proteins into secretory vesicles. At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane. There are multiple events that may occur between the budding of secretory vesicles from the Golgi and the fusion of these vesicles at the plasma membrane. The most obvious and best-known event is the transport of secretory vesicles from Golgi to the vicinity of the plasma membrane via microtubules and their associated motors. At the vicinity of the plasma membrane, however, it is not clear how vesicles finally dock and fuse with the plasma membrane. Identification of proteins involved in these events should provide important insights into the mechanisms of this little known stage of the exocytotic pathway. Currently, a protein complex, known as the sec6/8 or the exocyst complex, has been implicated to play a role at this late stage of exocytosis.

scholarly journals Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion

2001 ◽  
Vol 12 (11) ◽  
pp. 3690-3702 ◽  
Author(s):  
Naotada Ishihara ◽  
Maho Hamasaki ◽  
Sadaki Yokota ◽  
Kuninori Suzuki ◽  
Yoshiaki Kamada ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Secretory Pathway ◽  
De Novo ◽  
Subcellular Fractionation ◽  
Autophagosome Formation ◽  
Electron Microscopic ◽  
Membrane Flow ◽  
Protein Receptor ◽  
Sec Proteins ◽  
Protein Attachment

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeastSaccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (solubleN-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23,and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.

scholarly journals Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

2010 ◽  
Vol 188 (1) ◽  
pp. 145-156 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Benoît Zuber ◽  
Ulrike Elisabeth Maurer ◽  
Marek Cyrklaff ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Attachment Protein ◽  
Readily Releasable Pool ◽  
Protein Receptor ◽  
Membrane Contact ◽  
Synaptic Vesicle Release ◽  
Protein Attachment

The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.

scholarly journals The human SNARE protein Ykt6 mediates its own palmitoylation at C-terminal cysteine residues

2004 ◽  
Vol 384 (2) ◽  
pp. 233-237 ◽  
Author(s):  
Michael VEIT
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Covalent Attachment ◽  
Receptor Protein ◽  
Cysteine Residues ◽  
Snare Protein ◽  
Attachment Protein ◽  
Present Evidence ◽  
Protein Receptor ◽  
Protein Attachment

The yeast SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein Ykt6 was shown to mediate palmitoylation of the fusion factor Vac8 in a reaction essential for the fusion of vacuoles. Here I present evidence that hYkt6 (human Ykt6) has self-palmitoylating activity. Incubation of recombinant hYkt6 with [3H]Pal-CoA ([3H]palmitoyl-CoA) leads to covalent attachment of palmitate to C-terminal cysteine residues. The N-terminal domain of human Ykt6 contains a Pal-CoA binding site and is required for the reaction.

scholarly journals Snarepins Are Functionally Resistant to Disruption by Nsf and αSNAP

2000 ◽  
Vol 149 (5) ◽  
pp. 1063-1072 ◽  
Author(s):  
Thomas Weber ◽  
Francesco Parlati ◽  
James A. McNew ◽  
Robert J. Johnston ◽  
Benedikt Westermann ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Receptor Proteins ◽  
Protein Receptor ◽  
The Face ◽  
Target Membrane ◽  
The Moment ◽  
Protein Attachment ◽  
Lines Of Evidence

SNARE (SNAP [soluble NSF {N-ethylmaleimide–sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.

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