scholarly journals The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus

2008 ◽  
Vol 180 (4) ◽  
pp. 713-720 ◽  
Author(s):  
Auxiliadora Aguilera-Romero ◽  
Joanna Kaminska ◽  
Anne Spang ◽  
Howard Riezman ◽  
Manuel Muñiz
Keyword(s):  
Golgi Apparatus ◽  
Direct Evidence ◽  
Family Members ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Active Role ◽  
Small Gtpase ◽  
Vesicle Formation ◽  
Transport Vesicles ◽  
Cargo Proteins

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.

scholarly journals Biogenesis of γ-secretase early in the secretory pathway

2007 ◽  
Vol 179 (5) ◽  
pp. 951-963 ◽  
Author(s):  
Jinoh Kim ◽  
Bertrand Kleizen ◽  
Regina Choy ◽  
Gopal Thinakaran ◽  
Sangram S. Sisodia ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Evidence ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
Transient Intermediate ◽  
Important Enzyme

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.

scholarly journals Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

1998 ◽  
Vol 140 (3) ◽  
pp. 541-551 ◽  
Author(s):  
Harold D. Love ◽  
Chung-Chih Lin ◽  
Craig S. Short ◽  
Joachim Ostermann
Keyword(s):  
Golgi Apparatus ◽  
Functional Characterization ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Golgi Stack ◽  
Culture Cells ◽  
Golgi Membranes ◽  
Tissue Culture Cells ◽  
Transport Vesicles

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.

scholarly journals Determining the content of vesicles captured by golgin tethers using LOPIT-DC

2019 ◽  
Author(s):  
John J.H. Shin ◽  
Oliver M. Crook ◽  
Alicia Borgeaud ◽  
Jérôme Cattin-Ortolá ◽  
Sew-Yeu Peak-Chew ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Retrograde Transport ◽  
Statistical Modelling ◽  
Lysosomal Hydrolases ◽  
Transport Pathways ◽  
Transient Nature ◽  
Transport Vesicles ◽  
Cargo Proteins

AbstractThe internal organisation of the cell depends on tethers at destination organelles to selectively capture incoming transport vesicles to facilitate SNARE-mediated fusion. The golgin long coiled-coil proteins function as tethers that contributes to this specificity at the Golgi (1). Golgin-97, golgin-245 and GCC88 golgins of the trans-Golgi capture vesicles derived from endosomes, which serve to recycle the critical Golgi machinery required to deliver lysosomal hydrolases and to maintain exocytosis. Retrograde trafficking from endosomes to the trans-Golgi network (TGN) is a complex process that involves the sorting of transmembrane cargo proteins into distinct transport vesicles by adaptors from multiple pathways. The content of these distinct vesicles, which golgin they target and the factors that mediate this targeting are not well understood. The major challenges that have limited advances in these areas is the transient nature of vesicle tethering, and the redundancies in their mechanisms that confound experimental dissection. To gain better insight into these problems, we performed organelle proteomics using the Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) method on a system in which an ectopic golgin causes vesicles to accumulate in a tethered state (2). By incorporating Bayesian statistical modelling into our analysis (3), we determined that over 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network are on vesicles captured by golgin-97, including known cargo and components of the clathrin/AP-1, retromer-dependent and -independent transport pathways. We also determined a distinct class of vesicles shared by golgin-97, golgin-245 and GCC88 that is enriched in TMEM87A, a multi-pass transmembrane protein of unknown function that has previously been implicated in endosome-to-Golgi retrograde transport (4). Finally, we categorically demonstrate that the vesicles that these golgins capture are retrograde transport vesicles based on the lack of enrichment of lysosomal hydrolases in our LOPIT-DC data, and from correlative light electron tomography images of spherical vesicles captured by golgin-97. Together, our study demonstrates the power of combining LOPIT-DC with Bayesian statistical analysis in interrogating the dynamic spatial movement of proteins in transport vesicles.

scholarly journals The ArfGAP Glo3 Is Required for the Generation of COPI Vesicles

2004 ◽  
Vol 15 (9) ◽  
pp. 4064-4072 ◽  
Author(s):  
Stephen M. Lewis ◽  
Pak Phi Poon ◽  
Richard A. Singer ◽  
Gerald C. Johnston ◽  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Vesicular Transport ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Present Evidence ◽  
Gtpase Activating Proteins ◽  
Transport Vesicles ◽  
Negative Effect ◽  
Guanine Exchange Factors

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.

Vesicle formation in the Golgi apparatus

1989 ◽  
Vol 141 (4) ◽  
pp. 463-504 ◽  
Author(s):  
George F. Oster ◽  
Louis Y. Cheng ◽  
Hsiao-Ping H. Moore ◽  
Alan S. Perelson
Keyword(s):  
Golgi Apparatus ◽  
Vesicle Formation

scholarly journals Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

1998 ◽  
Vol 9 (12) ◽  
pp. 3561-3578 ◽  
Author(s):  
Harri Palokangas ◽  
Ming Ying ◽  
Kalervo Väänänen ◽  
Jaakko Saraste
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Marker Proteins ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Acidic Compartments

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

scholarly journals Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

2011 ◽  
Vol 22 (15) ◽  
pp. 2690-2701 ◽  
Author(s):  
Melissa D. Stuchell-Brereton ◽  
Amanda Siglin ◽  
Jun Li ◽  
Jeffrey K. Moore ◽  
Shubbir Ahmed ◽  
...  
Keyword(s):  
Light Chain ◽  
Direct Evidence ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Intermediate Chains ◽  
Activator Complex

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

Sign in / Sign up

Export Citation Format

Share Document