Concept: Endomembrane system
The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm -200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level.
As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.
Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease.
The vacuole is a prominent organelle that is essential for plant viability. The vacuole size, and its role in ion homeostasis, protein degradation and storage, place significant demands for trafficking of vacuolar cargo along the endomembrane system. Recent studies indicate that sorting of vacuolar cargo initiates at the ER and Golgi, but not the trans-Golgi network/early endosome, as previously thought. Furthermore, maturation of the trans-Golgi network into pre-vacuolar compartments seems to contribute to a major route for plant vacuolar traffic that works by bulk flow and ends with membrane fusion between the pre-vacuolar compartment and the tonoplast. Here we summarize recent evidence that indicates conserved and plant-specific mechanisms involved in sorting and trafficking of proteins to this major organelle.
Nuclear pore complexes (NPCs) are the sole gateway between the cytoplasm and the nucleus serving both as stringent permeability barrier and active transporters between the two compartments of eukaryotic cells. Complete mechanistic understanding of how these two functions are implemented within one and the same transport machine has not been attained to date. Based on several lines of structural evidence, a hypothesis was proposed postulating that NPCs shares common evolutionary origin with other intracellular systems responsible for active management of endomembranes. In this review we attempt to summarize the evidence supporting this hypothesis. The structural data obtained so far is evaluated and supplemented with the analysis of the functional evidence. Based on this analysis, a model is proposed which integrates the knowledge from the field of NPC function with that obtained from other endomembrane management systems in an attempt to shed new light on the mechanism of the NPC active transport.
The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.
Selective nuclear import in eukaryotic cells involves sequential interactions between nuclear import receptors and phenylalanine-glycine (FG)-repeat nucleoporins. Traditionally, binding of cargoes to import receptors is perceived as a nuclear pore complex independent event, while interactions between import complexes and nucleoporins are thought to take place at the nuclear pores. However, studies have shown that nucleoporins are mobile and not static within the nuclear pores, suggesting that they may become engaged in nuclear import prior to nuclear pore entry. Here we have studied post-mitotic nuclear import of the tumor suppressor protein PML. Since this protein forms nuclear compartments called PML bodies that persist during mitosis, the assembly of putative PML import complexes can be visualized on the surface of these protein aggregates as the cell progress from an import inactive state in mitosis to an import active state in G1. We show that these post-mitotic cytoplasmic PML bodies incorporate a multitude of peripheral nucleoporins, but not scaffold or nuclear basket nucleoporins, in a manner that depends on FG-repeats, the KPNB1 import receptor, and the PML nuclear localization signal. The study suggests that nucleoporins have the ability to target certain nuclear cargo proteins in a nuclear pore-uncoupled state, prior to nuclear pore entry.
The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside the sub-micrometer supramolecular complex. Here we combined super-resolution SPEED microscopy and nanobody specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveals both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provides evidence for their copy numbers of 8, 32, and 16 respectively per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes.
S-acylation/deacylation cycles and vesicular transport are critical for an adequate subcellular distribution of S-acylated Ras proteins. H-Ras is dually acylated on cysteines 181 and 184, but it is unknown how these residues individually contribute to H-Ras trafficking. In this study, we characterized the acylation and deacylation rates and membrane trafficking of mono-acylated H-Ras mutants to analyze their contributions to H-Ras plasma membrane and endomembrane distribution. We demonstrated that dually acylated H-Ras interacts with acyl-protein thioesterases (APT) 1 and 2 at the plasma membrane. Moreover, single acylation mutants of H-Ras differed not only in their subcellular distribution, where both proteins localized to different extents at both the Golgi complex and plasma membrane, but also in their deacylation rates, which we showed to be due to different sensitivities to APT1 and APT2. Fluorescence photobleaching and photoactivation experiments also revealed that 1) although S-acylated, single acylation mutants are incorporated with different efficiencies into Golgi complex to plasma membrane vesicular carriers and 2) the different deacylation rates of single acylated H-Ras influence differentially its overall exchange between different compartments by non-vesicular transport. Taken together, our results show that individual S-acylation sites provide singular information about H-Ras subcellular distribution that is required for GTPase signaling.
In this study, we carry out a systematic characterisation of the YIPF family of proteins with respect to their subcellular localisation profile, membrane topology and functional effects on the endomembrane system. YIPF proteins primarily localise to the Golgi complex and can be grouped into trans-Golgi-localising YIPFs (YIPF1 and YIPF2) and cis-Golgi-localising YIPFs (YIPF3, YIPF4 and YIPF5), with YIPF6 and YIPF7 showing a broader profile being distributed throughout the Golgi stack. YIPF proteins have a long soluble N-terminal region, which is orientated towards the cytosol, followed by 5 closely stacked transmembrane domains, and a C terminus, orientated towards the lumen of the Golgi. The significance of YIPF proteins for the maintenance of the morphology of the Golgi was tested by RNA interference, revealing a number of specific morphological changes to this organelle on their depletion. We propose a role for this family of proteins in regulating membrane dynamics in the endomembrane system.