Biogenesis of γ-secretase early in the secretory pathway

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.

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

Biochemical Journal ◽

10.1042/bj20070234 ◽

2007 ◽

Vol 409 (2) ◽

pp. 555-562 ◽

Cited By ~ 68

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.

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Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

10.17504/protocols.io.b3d9qi96 ◽

2022 ◽

Author(s):

Javier Manzano-Lopez†* ◽

Sofia Rodriguez-Gallardo† ◽

Susana Sabido-Bozo† ◽

Alejandro Cortes-Gomez ◽

Ana Maria Perez-Linero ◽

...

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Protein Complexes ◽

Secretory Protein ◽

Extracellular Proteins ◽

Transient Nature ◽

Transport Vesicles ◽

System P ◽

Cargo Receptor ◽

Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

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Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

10.17504/protocols.io.bytapwie ◽

2021 ◽

Author(s):

Javier Manzano-Lopez † ◽

Sofia Rodriguez-Gallardo † ◽

Susana Sabido-Bozo ◽

Ana Maria Perez-Linero ◽

Rafael Lucena ◽

...

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Protein Complexes ◽

Secretory Protein ◽

Extracellular Proteins ◽

Transient Nature ◽

Transport Vesicles ◽

System P ◽

Cargo Receptor ◽

Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

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The in silico human surfaceome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808790115 ◽

2018 ◽

Vol 115 (46) ◽

pp. E10988-E10997 ◽

Cited By ~ 44

Author(s):

Damaris Bausch-Fluck ◽

Ulrich Goldmann ◽

Sebastian Müller ◽

Marc van Oostrum ◽

Maik Müller ◽

...

Keyword(s):

Cell Surface ◽

In Silico ◽

Embryonic Stem ◽

Surface Protein ◽

Surface Proteins ◽

Cell Surface Protein ◽

Cell Surface Proteins ◽

A Cell ◽

Visualization Tools ◽

Protein Classes

Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.

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The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.

The Journal of Cell Biology ◽

10.1083/jcb.118.6.1321 ◽

1992 ◽

Vol 118 (6) ◽

pp. 1321-1332 ◽

Cited By ~ 31

Author(s):

B W Wattenberg ◽

T J Raub ◽

R R Hiebsch ◽

P J Weidman

Keyword(s):

Protein Transport ◽

Vesicle Formation ◽

Attachment Protein ◽

Direct Role ◽

Transport Vesicles ◽

Sensitive Factor ◽

Fusion Site ◽

Transport Vesicle ◽

Target Membrane ◽

A Cell

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.

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Functional correlation between cell adhesive properties and some cell surface proteins.

The Journal of Cell Biology ◽

10.1083/jcb.75.2.464 ◽

1977 ◽

Vol 75 (2) ◽

pp. 464-474 ◽

Cited By ~ 390

Author(s):

M Takeichi

Keyword(s):

Cell Surface ◽

Chinese Hamster ◽

Surface Protein ◽

Surface Proteins ◽

Adhesive Properties ◽

Cell Surface Proteins ◽

Morphological Studies ◽

Chinese Hamster V79 Cells ◽

A Cell ◽

In Cells

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+-dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+-dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.

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Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

10.1101/2021.07.28.454135 ◽

2021 ◽

Author(s):

Anna Cioce ◽

Beatriz Calle ◽

Andrea Marchesi ◽

Ganka Bineva-Todd ◽

Helen Flynn ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Surface ◽

Cell Line ◽

Specific Protein ◽

Surface Proteins ◽

Cancer Development ◽

Biological Processes ◽

Culture Studies ◽

Cell Surface Proteins ◽

A Cell

Interactions between cells fundamentally impact biological processes. In cancer development, such interactions define key stages of disease that cannot be adequately recapitulated in cell monoculture. Complex co-culture studies have been key to unraveling the complexity of these processes, usually by sorting cells and transcriptome or bulk proteome analyses. However, these methods invariably lead to sample loss and do not capture aberrant glycosylation as an important corollary of cancer formation. Here, we report the development of Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped with a biosynthetic AND gate that uses bioorthogonally tagged sugars to generate glycosylation precursors. The cellular glycosylation machinery then introduces bioorthogonal tags into glycoproteins exclusively in cell lines expressing the enzymes of the biosynthetic AND gate. Modification with clickable reporter moieties allows for imaging or enrichment with mass spectrometry-proteomics in a cell-specific fashion. Making use of glycans as a property of most cell surface proteins, we use BOCTAG as an efficient means for cell-specific protein tracing.

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Deubiquitinating enzymes Ubp2 and Ubp15 regulate endocytosis by limiting ubiquitination and degradation of ARTs

Molecular Biology of the Cell ◽

10.1091/mbc.e17-01-0008 ◽

2017 ◽

Vol 28 (9) ◽

pp. 1271-1283 ◽

Cited By ~ 18

Author(s):

Hsuan-Chung Ho ◽

Jason A. MacGurn ◽

Scott D. Emr

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Protein Composition ◽

Surface Proteins ◽

Endocytic Pathway ◽

Plasma Membrane Protein ◽

Deubiquitinating Enzymes ◽

Cell Surface Proteins ◽

Cargo Proteins ◽

The Stability

Endocytic down-regulation of cell-surface proteins is a fundamental cellular process for cell survival and adaptation to environmental stimuli. Ubiquitination of cargo proteins serves as the sorting signal for downstream trafficking and relies on the arrestin-related trafficking adaptor (ART)-Rsp5 ubiquitin ligase adaptor network in yeast. Hence proper regulation of the abundance and activity of these ligase–adaptor complexes is critical for maintenance of optimal plasma membrane protein composition. Here we report that the stability of ARTs is regulated by the deubiquitinating enzymes (DUBs) Ubp2 and Ubp15. By counteracting the E3 ubiquitin ligase Rsp5, Ubp2 and Ubp15 prevent hyperubiquitination and proteasomal degradation of ARTs. Specifically, we show that loss of both Ubp2 and Ubp15 results in a defect in Hxt6 endocytosis associated with Art4 instability. Our results uncover a novel function for DUBs in the endocytic pathway by which Ubp2 and Ubp15 positively regulate the ART-Rsp5 network.

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Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking

Journal of Virology ◽

10.1128/jvi.02741-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 3842-3851 ◽

Cited By ~ 74

Author(s):

Romuald Patient ◽

Christophe Hourioux ◽

Pierre-Yves Sizaret ◽

Sylvie Trassard ◽

Camille Sureau ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Secretory Pathway ◽

Surface Proteins ◽

Spherical Particles ◽

S Protein ◽

L Protein ◽

Transport Vesicles ◽

B Virus ◽

Virion Envelope

ABSTRACT Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.

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Protein sorting at the ER–Golgi interface

The Journal of Cell Biology ◽

10.1083/jcb.201610031 ◽

2016 ◽

Vol 215 (6) ◽

pp. 769-778 ◽

Cited By ~ 105

Author(s):

Natalia Gomez-Navarro ◽

Elizabeth Miller

Keyword(s):

Secretory Pathway ◽

Protein Sorting ◽

Vesicle Traffic ◽

Protein Traffic ◽

Cellular Physiology ◽

Transport Vesicles ◽

Cargo Proteins ◽

Vesicle Biogenesis ◽

Membrane Bound ◽

Insight Into

Protein traffic is of critical importance for normal cellular physiology. In eukaryotes, spherical transport vesicles move proteins and lipids from one internal membrane-bound compartment to another within the secretory pathway. The process of directing each individual protein to a specific destination (known as protein sorting) is a crucial event that is intrinsically linked to vesicle biogenesis. In this review, we summarize the principles of cargo sorting by the vesicle traffic machinery and consider the diverse mechanisms by which cargo proteins are selected and captured into different transport vesicles. We focus on the first two compartments of the secretory pathway: the endoplasmic reticulum and Golgi. We provide an overview of the complexity and diversity of cargo adaptor function and regulation, focusing on recent mechanistic discoveries that have revealed insight into protein sorting in cells.

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