scholarly journals Mechanism of Constitutive Export from the Golgi: Bulk Flow via the Formation, Protrusion, and En Bloc Cleavage of large trans-Golgi Network Tubular Domains

2003 ◽  
Vol 14 (11) ◽  
pp. 4470-4485 ◽  
Author(s):  
Elena V. Polishchuk ◽  
Alessio Di Pentima ◽  
Alberto Luini ◽  
Roman S. Polishchuk
Keyword(s):  
Three Dimensional ◽  
Bulk Flow ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
En Bloc ◽  
Trans Golgi Network ◽  
Membrane Fission ◽  
Large Structures ◽  
Cargo Proteins ◽  
Golgi Network

Transport of constitutive cargo proteins from the Golgi complex to the plasma membrane (PM) is known to be mediated by large tubular-saccular carriers moving along microtubules. However, the process by which these large structures emerge from the trans-Golgi network (TGN) remains unclear. Here, we address the question of the formation of Golgi-to-PM carriers (GPCs) by using a suitable cluster of morphological techniques, providing an integrated view of their dynamics and three-dimensional structure. Our results indicate that exit from the TGN of a constitutive traffic marker, the VSVG protein, occurs by bulk flow and is a three-step process. First, the formation of a tubular-reticular TGN domain (GPC precursor) that includes PM-directed proteins and excludes other cargo and Golgi-resident proteins. Notably, this step does not require membrane fusion. Second, the docking of this preformed domain on microtubules and its kinesin-mediated extrusion. Finally, the detachment of the extruded domain by membrane fission. The formation of GPCs does not involve cargo concentration and is not associated with the presence of known coat proteins on GPC precursors. In summary, export from the Golgi occurs via the formation, protrusion and en bloc cleavage of specialized TGN tubular-saccular domains.

scholarly journals Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast

2007 ◽  
Vol 18 (5) ◽  
pp. 1803-1815 ◽  
Author(s):  
Alenka Čopič ◽  
Trevor L. Starr ◽  
Randy Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Protein Transport ◽  
Adaptor Protein ◽  
Additional Function ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

scholarly journals 100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

1992 ◽  
Vol 117 (6) ◽  
pp. 1171-1179 ◽  
Author(s):  
DH Wong ◽  
FM Brodsky
Keyword(s):  
Membrane Binding ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Coated Pits ◽  
Binding Properties ◽  
Vitro Assay ◽  
Normal Cycle ◽  
Trans Golgi Network ◽  
Golgi Region ◽  
Golgi Network

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.

Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells

1990 ◽  
Vol 95 (3) ◽  
pp. 441-461
Author(s):  
G. Griffiths ◽  
R. Matteoni ◽  
R. Back ◽  
B. Hoflack
Keyword(s):  
Acid Phosphatase ◽  
Membrane Surface ◽  
Three Dimensional ◽  
Endocytic Pathway ◽  
Dimensional Structure ◽  
Surface Areas ◽  
Early Endosomes ◽  
Mannose 6 Phosphate ◽  
Golgi Network

The structure of a late endosomal compartment, which contains the bulk of the cation-independent mannose 6-phosphate receptor (MPR) in NRK cells, is documented using immunocytochemistry and cryo-sections, as well as conventional Epon sections. This compartment, which we refer to as the prelysosomal compartment (PLC), has a complex three-dimensional structure consisting of tubuloreticular domains in continuity with vesicular parts. The latter are characterized by a high density of internal membranes, which may be either tubular or sheet-like, that label extensively for the MPR. This structural organization was also maintained after fractionation in sucrose gradients. The amount of MPR immunolabelling was then quantitated with respect to the membrane surface areas of the four compartments where it is found: namely, the plasma membrane, early endosomes, the trans Golgi network and the PLC. The results showed that in NRK cells 90% of the labelling for the receptor was found in the PLC, with the rest distributed over the other three compartments. Cytochemical studies indicated that the PLC is the first structure along the endocytic pathway that gives a significant reaction for acid phosphatase. However, the PLC is clearly distinct from the MPR-negative lysosomes, which are also acid phosphatase-positive, since the two organelles could be physically separated from each other after fractionation on Percoll gradients.

scholarly journals The Structure of Bypass of Forespore C, an Intercompartmental Signaling Factor during Sporulation in Bacillus

2005 ◽  
Vol 280 (43) ◽  
pp. 36214-36220 ◽  
Author(s):  
Hayley M. Patterson ◽  
James A. Brannigan ◽  
Simon M. Cutting ◽  
Keith S. Wilson ◽  
Anthony J. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mother Cell ◽  
Asymmetric Cell Division ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Spore Coat Proteins ◽  
Α Helix ◽  
Β Sheet

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase σ-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing σK, (EσK). σK is synthesized as an inactive zymogen, pro-σK, which is activated by proteolytic cleavage. Processing of pro-σK is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-σK processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-σK processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded β-sheet onto one face of which an α-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded β-sheet and three α-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the σK checkpoint are discussed in the light of sequence and structure comparisons.

scholarly journals Three-dimensional Structure of Victorivirus HvV190S Suggests Coat Proteins in Most Totiviruses Share a Conserved Core

2013 ◽  
Vol 9 (3) ◽  
pp. e1003225 ◽  
Author(s):  
Sarah E. Dunn ◽  
Hua Li ◽  
Giovanni Cardone ◽  
Max L. Nibert ◽  
Said A. Ghabrial ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
Three Dimensional Structure ◽  
Conserved Core

scholarly journals Three-dimensional structure of 22 uncultured ssRNA bacteriophages: Flexibility of the coat protein fold and variations in particle shapes

2020 ◽  
Vol 6 (36) ◽  
pp. eabc0023
Author(s):  
Jānis Rūmnieks ◽  
Ilva Liekniņa ◽  
Gints Kalniņš ◽  
Mihails Šišovs ◽  
Ināra Akopjana ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Binding Mode ◽  
Structural Proteins ◽  
Mutation Rates ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
Protein Fold ◽  
Three Dimensional Structure ◽  
Dsrna Binding ◽  
Particle Shapes

The single-stranded RNA (ssRNA) bacteriophages are among the simplest known viruses with small genomes and exceptionally high mutation rates. The number of ssRNA phage isolates has remained very low, but recent metagenomic studies have uncovered an immense variety of distinct uncultured ssRNA phages. The coat proteins (CPs) in these genomes are particularly diverse, with notable variation in length and often no recognizable similarity to previously known viruses. We recombinantly expressed metagenome-derived ssRNA phage CPs to produce virus-like particles and determined the three-dimensional structure of 22 previously uncharacterized ssRNA phage capsids covering nine distinct CP types. The structures revealed substantial deviations from the previously known ssRNA phage CP fold, uncovered an unusual prolate particle shape, and revealed a previously unseen dsRNA binding mode. These data expand our knowledge of the evolution of viral structural proteins and are of relevance for applications such as ssRNA phage–based vaccine design.

scholarly journals Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (5) ◽  
Author(s):  
Xiao Tang ◽  
Feng Yang ◽  
Yusong Guo
Keyword(s):  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Golgi Network

scholarly journals Characterization of a Fourth Adaptor-related Protein Complex

1999 ◽  
Vol 10 (8) ◽  
pp. 2787-2802 ◽  
Author(s):  
Jennifer Hirst ◽  
Nicholas A. Bright ◽  
Brian Rous ◽  
Margaret S. Robinson
Keyword(s):  
Expressed Sequence Tag ◽  
Protein Complexes ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Cell Types ◽  
Small Gtpase ◽  
Immunogold Electron Microscopy ◽  
Coat Proteins ◽  
Cargo Proteins ◽  
Golgi Network

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Sign in / Sign up

Export Citation Format

Share Document