Illuminating the secretory pathway: when do we need vesicles?

2001 ◽  
Vol 114 (6) ◽  
pp. 1053-1059 ◽  
Author(s):  
D.J. Stephens ◽  
R. Pepperkok
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Time Lapse ◽  
Membrane Structures ◽  
Long Distance ◽  
Transport Pathways ◽  
Transport Vesicles ◽  
Range Transport

Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300–500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60–80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane. http://www.biologists.com/JCS/movies/jcs2220.html

scholarly journals ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

1999 ◽  
Vol 147 (7) ◽  
pp. 1457-1472 ◽  
Author(s):  
Chung-Chih Lin ◽  
Harold D. Love ◽  
Jennifer N. Gushue ◽  
John J.M. Bergeron ◽  
Joachim Ostermann
Keyword(s):  
Secretory Pathway ◽  
Mutant Cell ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Intermediate Step ◽  
Golgi Stack ◽  
Transport Vesicles ◽  
Direct Fusion

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3623-3633 ◽  
Author(s):  
J. Jantti ◽  
S. Keranen ◽  
J. Toikkanen ◽  
E. Kuismanen ◽  
C. Ehnholm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Signal Sequence ◽  
Insertion Site ◽  
Membrane Insertion ◽  
Membrane Anchor

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

The trials and tubule-ations of Rab6 involvement in Golgi-to-ER retrograde transport

2014 ◽  
Vol 42 (5) ◽  
pp. 1453-1459 ◽  
Author(s):  
Linda F. Heffernan ◽  
Jeremy C. Simpson
Keyword(s):  
Secretory Pathway ◽  
Size Composition ◽  
Retrograde Transport ◽  
Retrograde Flow ◽  
Transport Pathway ◽  
Membrane Flow ◽  
Transport Pathways ◽  
Complex Needs ◽  
Early Secretory Pathway ◽  
Regulatory Machinery

In the early secretory pathway, membrane flow in the anterograde direction from the endoplasmic reticulum (ER) to the Golgi complex needs to be tightly co-ordinated with retrograde flow to maintain the size, composition and functionality of these two organelles. At least two mechanisms of transport move material in the retrograde direction: one regulated by the cytoplasmic coatomer protein I complex (COPI), and a second COPI-independent pathway utilizing the small GTP-binding protein Rab6. Although the COPI-independent pathway was discovered 15 years ago, it remains relatively poorly characterized, with only a handful of machinery molecules associated with its operation. One feature that makes this pathway somewhat unusual, and potentially difficult to study, is that the transport carriers predominantly seem to be tubular rather than vesicular in nature. This suggests that the regulatory machinery is likely to be different from that associated with vesicular transport pathways controlled by conventional coat complexes. In the present mini-review, we have highlighted the key experiments that have characterized this transport pathway so far and also have discussed the challenges that lie ahead with respect to its further characterization.

scholarly journals Structural Diversities of Lectins Binding to the Glycosphingolipid Gb3

2021 ◽  
Vol 8 ◽  
Author(s):  
Lina Siukstaite ◽  
Anne Imberty ◽  
Winfried Römer
Keyword(s):  
Plasma Membrane ◽  
Retrograde Transport ◽  
Initial Step ◽  
Cell Receptor ◽  
Membrane Curvature ◽  
Amino Acid Sequences ◽  
Shigella Dysenteriae ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Protein Receptors

Glycolipids are present on the surfaces of all living cells and thereby represent targets for many protein receptors, such as lectins. Understanding the interactions between lectins and glycolipids is essential for investigating the functions of lectins and the dynamics of glycolipids in living membranes. This review focuses on lectins binding to the glycosphingolipid globotriaosylceramide (Gb3), an attractive host cell receptor, particularly for pathogens and pathogenic products. Shiga toxin (Stx), from Shigella dysenteriae or Escherichia coli, which is one of the most virulent bacterial toxins, binds and clusters Gb3, leading to local negative membrane curvature and the formation of tubular plasma membrane invaginations as the initial step for clathrin-independent endocytosis. After internalization, it is embracing the retrograde transport pathway. In comparison, the homotetrameric lectin LecA from Pseudomonas aeruginosa can also bind to Gb3, triggering the so-called lipid zipper mechanism, which results in membrane engulfment of the bacterium as an important step for its cellular uptake. Notably, both lectins bind to Gb3 but induce distinct plasma membrane domains and exploit mainly different transport pathways. Not only, several other Gb3-binding lectins have been described from bacterial origins, such as the adhesins SadP (from Streptococcus suis) and PapG (from E. coli), but also from animal, fungal, or plant origins. The variety of amino acid sequences and folds demonstrates the structural versatilities of Gb3-binding lectins and asks the question of the evolution of specificity and carbohydrate recognition in different kingdoms of life.

scholarly journals Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (8) ◽  
pp. 3243-3255 ◽  
Author(s):  
Geng Wang ◽  
Robert J. Deschenes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Ras Proteins ◽  
Subcellular Targeting ◽  
Transport Pathways ◽  
Mitochondrial Defects ◽  
Protease Treatment ◽  
Class C

ABSTRACT Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.

scholarly journals Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

1999 ◽  
Vol 341 (2) ◽  
pp. 323-327 ◽  
Author(s):  
Mark R. JACKMAN ◽  
Juliet A. ELLIS ◽  
Sally R. GRAY ◽  
Wenda SHURETY ◽  
J. Paul LUZIO
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Mammalian Cells ◽  
Cell Clone ◽  
Membrane Traffic ◽  
Retrograde Transport ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Cell Polarization ◽  
Apical Plasma Membrane

It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

scholarly journals FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

2010 ◽  
Vol 21 (17) ◽  
pp. 3041-3053 ◽  
Author(s):  
Jian Jing ◽  
Jagath R. Junutula ◽  
Christine Wu ◽  
Jemima Burden ◽  
Hugo Matern ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Recycling Endosome ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Transport Vesicles ◽  
Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

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 Misfolded GPI-anchored proteins are escorted through the secretory pathway by ER-derived factors

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eszter Zavodszky ◽  
Ramanujan S Hegde
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Quantitative Proteomics ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Flow Cytometry Analysis ◽  
Time Resolved ◽  
Interaction Partners ◽  
Quantitative Flow

We have used misfolded prion protein (PrP*) as a model to investigate how mammalian cells recognize and degrade misfolded GPI-anchored proteins. While most misfolded membrane proteins are degraded by proteasomes, misfolded GPI-anchored proteins are primarily degraded in lysosomes. Quantitative flow cytometry analysis showed that at least 85% of PrP* molecules transiently access the plasma membrane en route to lysosomes. Unexpectedly, time-resolved quantitative proteomics revealed a remarkably invariant PrP* interactome during its trafficking from the endoplasmic reticulum (ER) to lysosomes. Hence, PrP* arrives at the plasma membrane in complex with ER-derived chaperones and cargo receptors. These interaction partners were critical for rapid endocytosis because a GPI-anchored protein induced to misfold at the cell surface was not recognized effectively for degradation. Thus, resident ER factors have post-ER itineraries that not only shield misfolded GPI-anchored proteins during their trafficking, but also provide a quality control cue at the cell surface for endocytic routing to lysosomes.

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