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

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.

Biochemical and Cytological Interactions Between Callose Synthase and Microtubules in the Tobacco Pollen Tube

Callose is a cell wall polysaccharide involved in several fundamental biological processes, ranging from plant development to response to abiotic and biotic stresses. To understand how callose deposition is regulated, it is important to know how its synthesizing enzyme, i.e., callose synthase, is regulated and if it interacts with vesicular-cytoskeletal system of plant cells. Actin filaments are thought to determine the long-range distribution of callose synthase through transport vesicles. Unlike other enzymes (such as cellulose synthase) that synthesize cell wall polysaccharides, the spatial and biochemical relationships between callose synthase and microtubules are poorly understood. Some experimental evidence already support the association between callose synthase and tubulin, however, despite its importance in maintaining plant integrity, knowledge about regulation of callose biosynthesis is still limited. Here we investigated the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in a model system, pollen tube, where callose is an essential cell wall component. Native 2-D electrophoresis and isolation of the callose synthase complex confirmed that callose synthase is associated with tubulin and can interface with cortical microtubules. In contrast, actin and sucrose synthase (which supplies UDP-glucose to callose synthase) are not permanently associated with callose synthase. Immunogold labeling showed strong colocalization of the enzyme and microtubules; this association is occasionally mediated by vesicles. The association between callose synthase and vesicles was also demonstrated by co-distribution between the enzyme and Rab11b; in addition, the not homogeneous distribution of callose synthase in cell membranes is also shown by analysis of membrane microdomains.

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

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.

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

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.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v2

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.

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

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.

GOLPH3 keeps the Golgi residents at home

In this issue of JCB, Welch et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

The fidelity of Golgi glycosylation is, in part, ensured by compartmentalization of enzymes within the stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterization of the clientele of GOLPH3 and its paralogue GOLPH3L is lacking. GOLPH3’s role is of particular interest as it is frequently amplified in several solid tumor types. Here, we apply two orthogonal proteomic methods to identify GOLPH3+3L clients and find that they act in diverse glycosylation pathways or have other roles in the Golgi. Binding studies, bioinformatics, and a Golgi retention assay show that GOLPH3+3L bind the cytoplasmic tails of their clients through membrane-proximal positively charged residues. Furthermore, deletion of GOLPH3+3L causes multiple defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

Non-Heating Alternating Magnetic Field Nanomechanical Stimulation of Biomolecule Structures via Magnetic Nanoparticles as the Basis for Future Low-Toxic Biomedical Applications

The review discusses the theoretical, experimental and toxicological aspects of the prospective biomedical application of functionalized magnetic nanoparticles (MNPs) activated by a low frequency non-heating alternating magnetic field (AMF). In this approach, known as nano-magnetomechanical activation (NMMA), the MNPs are used as mediators that localize and apply force to such target biomolecular structures as enzyme molecules, transport vesicles, cell organelles, etc., without significant heating. It is shown that NMMA can become a biophysical platform for a family of therapy methods including the addressed delivery and controlled release of therapeutic agents from transport nanomodules, as well as selective molecular nanoscale localized drugless nanomechanical impacts. It is characterized by low system biochemical and electromagnetic toxicity. A technique of 3D scanning of the NMMA region with the size of several mm to several cm over object internals has been described.