scholarly journals Evidence for Segregation of Sphingomyelin and Cholesterol during Formation of Copi-Coated Vesicles

2000 ◽  
Vol 151 (3) ◽  
pp. 507-518 ◽  
Author(s):  
Britta Brügger ◽  
Roger Sandhoff ◽  
Sabine Wegehingel ◽  
Karin Gorgas ◽  
Jörg Malsam ◽  
...  
Keyword(s):  
Cholesterol Synthesis ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Lipid Analysis ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
A Cell ◽  
Higher Eukaryotes ◽  
Specific Lipid

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

scholarly journals Erv41p and Erv46p

2001 ◽  
Vol 152 (3) ◽  
pp. 503-518 ◽  
Author(s):  
Stefan Otte ◽  
William J. Belden ◽  
Matthew Heidtman ◽  
Jay Liu ◽  
Ole N. Jensen ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Cold Sensitivity ◽  
Ionization Mass ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Yeast Strains ◽  
Mutant Strains ◽  
A Cell ◽  
Copii Vesicles ◽  
Vesicle Proteins

Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Δ strain, and Erv41p was not detected in an erv46Δ strain. When the erv41Δ or ev46Δ alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p–Erv46p complex influences the membrane fusion stage of transport.

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

2014 ◽  
Vol 79 (9) ◽  
pp. 879-893 ◽  
Author(s):  
A. I. Fokin ◽  
I. B. Brodsky ◽  
A. V. Burakov ◽  
E. S. Nadezhdina
Keyword(s):  
Secretory Pathway ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Microtubule Motors

scholarly journals Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

2020 ◽  
Vol 133 (19) ◽  
pp. jcs250100
Author(s):  
Simon Newstead ◽  
Francis Barr
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Protein Localisation ◽  
And Function ◽  
Trafficking Receptor

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

scholarly journals Novel Isotypic γ/ζ Subunits Reveal Three Coatomer Complexes in Mammals

2004 ◽  
Vol 24 (3) ◽  
pp. 1070-1080 ◽  
Author(s):  
Dominik Wegmann ◽  
Pablo Hess ◽  
Carola Baier ◽  
Felix T. Wieland ◽  
Constanze Reinhard
Keyword(s):  
Coat Protein ◽  
Secretory Pathway ◽  
Small Gtpase ◽  
Liver Cytosol ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Secretory Transport ◽  
Adp Ribosylation Factor ◽  
Precise Role

ABSTRACT In early secretory transport, coat recruitment for the formation of coat protein I (COPI) vesicles involves binding to donor Golgi membranes of the small GTPase ADP-ribosylation factor 1 and subsequent attachment of the cytoplasmic heptameric complex coatomer. Various hypotheses exist as to the precise role of and possible routes taken by COPI vesicles in the mammalian cell. Here we report the ubiquitous expression of two novel isotypes of coatomer subunits γ- and ζ-COP that are incorporated into coatomer, and show that three isotypes exist of the complex defined by the subunit combinations γ1/ζ1, γ1/ζ2, and γ2/ζ1. In a liver cytosol, these forms make up the total coatomer in a ratio of about 2:1:2, respectively. The coatomer isotypes are located differentially within the early secretory pathway, and the γ2/ζ1 isotype is preferentially incorporated into COPI vesicles. A population of COPI vesicles was characterized that almost exclusively contains γ2/ζ1 coatomer. This existence of three structurally different forms of coatomer will need to be considered in future models of COPI-mediated transport.

scholarly journals Auxilin facilitates membrane traffic in the early secretory pathway

2016 ◽  
Vol 27 (1) ◽  
pp. 127-136 ◽  
Author(s):  
Jingzhen Ding ◽  
Verónica A. Segarra ◽  
Shuliang Chen ◽  
Huaqing Cai ◽  
Sandra K. Lemmon ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Complexes ◽  
Coated Vesicles ◽  
Genetic Approach ◽  
Vesicle Budding ◽  
Early Secretory Pathway ◽  
Transport Vesicles ◽  
Copii Vesicles ◽  
Mutant Cells ◽  
Analogous Function

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER–Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

scholarly journals MicroRNAs and long non-coding RNAs as novel regulators of ribosome biogenesis

2020 ◽  
Vol 48 (2) ◽  
pp. 595-612 ◽  
Author(s):  
Mason A. McCool ◽  
Carson J. Bryant ◽  
Susan J. Baserga
Keyword(s):  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Ribosomal Subunits ◽  
Additional Layer ◽  
Disease Outcomes ◽  
A Cell ◽  
Non Coding Rnas ◽  
Higher Eukaryotes ◽  
Pathological Disease

Ribosome biogenesis is the fine-tuned, essential process that generates mature ribosomal subunits and ultimately enables all protein synthesis within a cell. Novel regulators of ribosome biogenesis continue to be discovered in higher eukaryotes. While many known regulatory factors are proteins or small nucleolar ribonucleoproteins, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) are emerging as a novel modulatory layer controlling ribosome production. Here, we summarize work uncovering non-coding RNAs (ncRNAs) as novel regulators of ribosome biogenesis and highlight their links to diseases of defective ribosome biogenesis. It is still unclear how many miRNAs or lncRNAs are involved in phenotypic or pathological disease outcomes caused by impaired ribosome production, as in the ribosomopathies, or by increased ribosome production, as in cancer. In time, we hypothesize that many more ncRNA regulators of ribosome biogenesis will be discovered, which will be followed by an effort to establish connections between disease pathologies and the molecular mechanisms of this additional layer of ribosome biogenesis control.

scholarly journals Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

2011 ◽  
Vol 286 (18) ◽  
pp. 16363-16373 ◽  
Author(s):  
Ayako Fukunaka ◽  
Yayoi Kurokawa ◽  
Fumie Teranishi ◽  
Israel Sekler ◽  
Kimimitsu Oda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Zinc Content ◽  
Protein Stabilization ◽  
Zinc Transport ◽  
Early Secretory Pathway ◽  
Tissue Nonspecific Alkaline Phosphatase ◽  
Dt40 Cells ◽  
Step Mechanism

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

scholarly journals The transmembrane protein p23 contributes to the organization of the Golgi apparatus

2000 ◽  
Vol 113 (6) ◽  
pp. 1043-1057 ◽  
Author(s):  
M. Rojo ◽  
G. Emery ◽  
V. Marjomaki ◽  
A.W. McDowall ◽  
R.G. Parton ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Ectopic Expression ◽  
Retrograde Transport ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Constant Diameter ◽  
Golgi Network

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

scholarly journals Targeting CCR5 trafficking to inhibit HIV-1 infection

2019 ◽  
Vol 5 (10) ◽  
pp. eaax0821 ◽  
Author(s):  
Gaelle Boncompain ◽  
Floriane Herit ◽  
Sarah Tessier ◽  
Aurianne Lescure ◽  
Elaine Del Nery ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Molecular Level ◽  
High Content Screening ◽  
Differential Protein ◽  
Early Secretory Pathway ◽  
A Cell ◽  
Hiv Entry ◽  
Hiv 1

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.

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