The N-region sequence context impacts the chloroplast import efficiency of multi-TMD protein.

Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region's role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.

Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy.

Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy.

An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex.

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and ß2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.

Rapamycin-Induced Translocation of Meiotic Nuclear Proteins in Saccharomyces cerevisiae.

Conditional depletion of proteins is a potential strategy to elucidate protein function, especially in complex cellular processes like meiosis. Several methods are available to effectively deplete a protein in a conditional manner. Conditional loss of a protein function can be achieved by depleting it from its region of action by degrading it. A conditional loss of protein function can also be achieved by sequestering it to a functionally unavailable compartment inside the cell. This chapter describes anchor away, a conditional depletion tool that can deplete a protein both temporally and spatially by translocation. It utilizes the affinity of FRB to bind FKBP12 in the presence of rapamycin for a quick and efficient translocation of the protein to a designated location. Anchor away is a reliable tool for the study of meiotic proteins, as only small quantities of rapamycin are required to efficiently and rapidly translocate the protein of interest without compromising meiotic progression.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

The nonphototrophic hypocotyl 3 (NPH3) domain protein NRL5 is a trafficking-associated GTPase essential for drought resistance.

The mechanisms of plant drought resistance are unclear but may involve membrane trafficking and metabolic reprogramming, including proline accumulation. Forward genetic screening using a proline dehydrogenase 1 (ProDH1) promoter:reporter identified a drought hypersensitive mutant with a single-amino acid substitution (P335L) in the nonphototrophic hypocotyl 3 (NPH3) domain of NPH3/root phototropism 2-like 5 (NRL5)/naked pins in Yucca 8 (NPY8). Further experiments found that NRL5 and other NPH3 domain proteins are guanosine triphosphatases (GTPases). NRL5, but not NRL5P335L, interacted with the RABE1c and RABH1b GTPases and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Vesicle-Associated Membrane Protein (VAMP)721/722. These proteins controlled NRL5 localization and connection to trafficking while also being genetically downstream of, and potentially regulated by, NRL5. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also reveal unexpected functions of the NPH3 domain such that the role of NPH3 domain proteins in signaling, trafficking, and cellular polarity can be critically reevaluated.

IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport.

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.

Conventional and Unconventional Protein Secretion in Yeast and Animal Cells.

Protein secretion mediated by the secretory transport pathway is a sophisticated and highly regulated cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized proteins pass through several endomembrane compartments to reach their destinations. This transport occurs via small, membrane-enclosed vesicles. To ensure the fidelity of trafficking, eukaryotic cells employ elaborate molecular machinery to accurately sort newly synthesized proteins into specific transport vesicles and precisely deliver them to respective acceptor compartments. Leaderless cargo proteins, lacking a signal peptide, follow an unconventional secretory pathway. This review encompasses the molecular machinery regulating both conventional and unconventional protein secretion in yeast and animal cells.

Bioinformatic Analysis for Identifying Transcription Factors Involved in Protein Secretion and Vacuole Formation.

Protein secretion and vacuole formation are vital processes in plant cells, playing crucial roles in various aspects of plant development, growth, and stress responses. Multiple regulators have been uncovered to be involved in these processes. In animal cells, the transcription factor TFEB has been extensively studied and its role in lysosomal biogenesis is well understood. However, the transcription factors governing protein secretion and vacuole formation in plants remain largely unexplored. In recent years, an increasing number of bioinformatics databases and tools have been developed, facilitating computational prediction and analysis of the function of genes or proteins in specific cellular processes. Leveraging these resources, this chapter aims to provide practical guidance on how to effectively utilize these existing databases and tools for the analysis of key transcription factors involved in regulating protein secretion and vacuole formation in plants, with a particular focus on Arabidopsis and other higher plants. The findings from this analysis can serve as a valuable resource for future experimental investigations and the development of targeted strategies to manipulate protein secretion and vacuole formation in plants.

An Overview of Protein Secretion in Plant Cells.

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants.

Quantifying Protein Dynamics by Kymograph Analysis.

Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.

Utilizing Plant Protein Secretion System for Pharmaceutical Protein Production.

Molecular farming, also known as plant molecular farming (PMF), is a technique that involves using plants and plant cells as bioreactors to produce recombinant proteins. This is a cost-effective and sustainable way of producing large quantities of proteins for various applications, including pharmaceuticals, vaccines, and industrial enzymes. An endogenous or exogenous signal peptide (SP) is flanked at the N-terminal for recombinant protein targeting and storage. These SPs are responsible for guiding the recombinant protein products to the correct destination within the plant cell or facilitating their secretion into the extracellular space. In this chapter, we will give a brief introduction of the current PMF research outcomes supported by the basic study of vesicle trafficking and protein secretion, mainly introducing the bright yellow 2 (BY-2) cell-based secretion pathway and its associated protocols according to our study of recombinant human iduronidase.

Identification of Neighboring Proteins of Endosomal Regulators by Using TurboID-Based Proximity Labeling.

In the endomembrane system, multivesicular bodies (MVBs) play a crucial role in sorting ubiquitinated membrane proteins into intraluminal vesicles for degradation upon fusion with vacuoles or lysosomes. This process involves regulations by multiprotein complexes, including endosomal sorting complex required for transport (ESCRT) I-III, and accessory proteins. Although many organellar proteomes have been identified in plant cells, the information of specific proteomes associated with regulators engaged in MVB biogenesis remains limited. Here, using the ESCRT component FREE1 as an example, we describe a method to identify neighboring proteins of endosomal regulators by using an approach of TurboID-based proximity labeling.

Pharmaceutical Analysis in the Plant Endomembrane System.

Vesicle trafficking is an essential cellular process conserved in eukaryotes to precisely transport proteins to their destinations. The plant endomembrane system plays a pivotal role in orchestrating this vesicle-mediated protein transport process, making its study essential for a comprehensive understanding of plant growth and development. Pharmaceutical analysis proves highly useful in investigating the plant endomembrane system. To facilitate further studies in this area, we present a summary of several commonly used chemical inhibitors in this chapter, providing a practical resource for researchers interested in the plant endomembrane system.

Genetic Screening of Factors in the Plant Protein Secretion.

The endomembrane system in plants is composed of interconnected membrane organelles that contribute to intracellular structure and function. These organelles include the endoplasmic reticulum (ER), Golgi apparatus, vacuole, trans-Golgi network, and prevacuolar compartment or multivesicular body. Through vesicle-mediated transport, secreted proteins are synthesized in the ER and subsequently transported along the secretory pathway to the vacuole or outside of cells to fulfill specialized functions. Genetic screening is a crucial method for studying plant protein secretion. It entails identifying phenotypic differences resulting from genetic mutations, such as ethyl methanesulfonate, T-DNA insertion, and RNAi, to investigate gene function and discover mutants with specific traits or gene functions. Significant progress has been achieved in the study of plant protein secretion through genetic screening. In this protocol, we provide a step-by-step guide to studying the protein secretion pathway using a genetic screen approach. We use the example of the free 1 suppressor of Arabidopsis thaliana and oil body mutants of Marchantia polymorpha. Additionally, we offer an overview of genetic screening and briefly summarize the emerging technologies in the field of protein secretion research.

Contribution of intraflagellar transport to compartmentalization and maintenance of the photoreceptor cell.

The first steps of vision take place in the ciliary outer segment compartment of photoreceptor cells. The protein composition of outer segments is uniquely suited to perform this function. The most abundant among these proteins is the visual pigment, rhodopsin, whose outer segment trafficking involves intraflagellar transport (IFT). Here, we report three major findings from the analysis of mice in which ciliary transport was acutely impaired by conditional knockouts of IFT-B subunits. First, we demonstrate the existence of a sorting mechanism whereby mislocalized rhodopsin is recruited to and concentrated in extracellular vesicles prior to their release, presumably to protect the cell from adverse effects of protein mislocalization. Second, reducing rhodopsin expression significantly delays photoreceptor degeneration caused by IFT disruption, suggesting that controlling rhodopsin levels may be an effective therapy for some cases of retinal degenerative disease. Last, the loss of IFT-B subunits does not recapitulate a phenotype observed in mutants of the BBSome (another ciliary transport protein complex relying on IFT) in which non-ciliary proteins accumulate in the outer segment. Whereas it is widely thought that the role of the BBSome is to primarily participate in ciliary transport, our data suggest that the BBSome has another major function independent of IFT and possibly related to maintaining the diffusion barrier of the ciliary transition zone.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Insulin-Activated Signaling Pathway and GLUT4 Membrane Translocation in hiPSC-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a cell model now widely used to investigate pathophysiological features of cardiac tissue. Given the invaluable contribution hiPSC-CM could make for studies on cardio-metabolic disorders by defining a postnatal metabolic phenotype, our work herein focused on monitoring the insulin response in CM derived from the hiPSC line UKBi015-B. Western blot analysis on total cell lysates obtained from hiPSC-CM showed increased phosphorylation of both AKT and AS160 following insulin treatment, but failed to highlight any changes in the expression dynamics of the glucose transporter GLUT4. By contrast, the Western blot analysis of membrane fractions, rather than total lysates, revealed insulin-induced plasma membrane translocation of GLUT4, which is known to also occur in postnatal CM. Thus, these findings suggest that hiPSC-derived CMs exhibit an insulin response reminiscent to that of adult CMs regarding intracellular signaling and GLUT4 translocation to the plasma membrane, representing a suitable cellular model in the cardio-metabolic research field. Moreover, our studies also demonstrate the relevance of analyzing membrane fractions rather than total lysates in order to monitor GLUT4 dynamics in response to metabolic regulators in hiPSC-CMs.

The endolysosomal system in conventional and unconventional protein secretion.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Mitochondrial-derived compartments remove surplus proteins from the outer mitochondrial membrane.

The outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a mitochondrial remodeling mechanism called the mitochondrial-derived compartment (MDC) that removes a subset of the mitochondrial proteome. Here, we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are incorporated into MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface.