scholarly journals Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Julia P Steringer ◽  
Sascha Lange ◽  
Sabína Čujová ◽  
Radek Šachl ◽  
Chetan Poojari ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Translocation ◽  
Membrane Translocation ◽  
Unconventional Secretion ◽  
Molecular Machinery ◽  
A Subunit ◽  
Heparan Sulfates ◽  
Dynamics Simulations ◽  
Key Steps ◽  
Inside Out

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.

scholarly journals Single event visualization of unconventional secretion of FGF2

2018 ◽  
Vol 218 (2) ◽  
pp. 683-699 ◽  
Author(s):  
Eleni Dimou ◽  
Katia Cosentino ◽  
Evgenia Platonova ◽  
Uris Ros ◽  
Mohsen Sadeghi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Living Cells ◽  
Membrane Translocation ◽  
Membrane Recruitment ◽  
Membrane Pores ◽  
Simultaneous Imaging ◽  
Unconventional Secretion ◽  
Heparan Sulfates ◽  
Kinetics Of ◽  
Outer Plasma Membrane

FGF2 is exported from cells by an unconventional secretory mechanism. Here, we directly visualized individual FGF2 membrane translocation events at the plasma membrane using live cell TIRF microscopy. This process was dependent on both PI(4,5)P2–mediated recruitment of FGF2 at the inner leaflet and heparan sulfates capturing FGF2 at the outer plasma membrane leaflet. By simultaneous imaging of both FGF2 membrane recruitment and the appearance of FGF2 at the cell surface, we revealed the kinetics of FGF2 membrane translocation in living cells with an average duration of ∼200 ms. Furthermore, we directly demonstrated FGF2 oligomers at the inner leaflet of living cells with a FGF2 dimer being the most prominent species. We propose this dimer to represent a key intermediate in the formation of higher FGF2 oligomers that form membrane pores and put forward a kinetic model explaining the mechanism by which membrane-inserted FGF2 oligomers serve as dynamic translocation intermediates during unconventional secretion of FGF2.

scholarly journals Archaeal and Bacterial SecD and SecF Homologs Exhibit Striking Structural and Functional Conservation

2006 ◽  
Vol 188 (4) ◽  
pp. 1251-1259 ◽  
Author(s):  
Nicholas J. Hand ◽  
Reinhard Klein ◽  
Anke Laskewitz ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Translocation ◽  
Specific Protein ◽  
Cold Sensitivity ◽  
Secretory Proteins ◽  
E Coli ◽  
Functional Conservation ◽  
Membrane Complex ◽  
Hydrophobic Membranes ◽  
A Subunit ◽  
Native Host

ABSTRACT The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii ΔsecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

scholarly journals Translocation of a Polymer through a Crowded Channel under Electrical Force

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Tingting Sun ◽  
Yunxin Gen ◽  
Hujun Xie ◽  
Zhouting Jiang ◽  
Zhiyong Yang
Keyword(s):  
Field Strength ◽  
Shape Factor ◽  
Cylindrical Channel ◽  
Protein Translocation ◽  
Scaling Exponent ◽  
Scaling Relation ◽  
Maximum Value ◽  
Translocation Time ◽  
Dynamics Simulations ◽  
Number Of Segments

The translocation of a polymer chain through a crowded cylindrical channel is studied using the Langevin dynamics simulations. The influences of the field strength F, the chain length N, and the crowding extent ρ on the translocation time are evaluated, respectively. Scaling relation τ~F-α is observed. With the crowding extent ρ increasing, the scaling exponent α becomes large. It is found that, for noncrowded channel, translocation probability drops when the field strength becomes large. However, for high-crowded channel, it is the opposite. Moreover, the translocation time and the average translocation time for all segments both have exponential growth with the crowding extent. The investigation of shape factor δ shows maximum value with increasing of the number of segments outside s. At last, the number of segments inside channel Nin in the process of translocation is calculated and a peak is observed. All the information from the study may benefit protein translocation.

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