ATPase in mature and differentiating phloem and xylem

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.     

Chlamydiae Assemble a Pathogen Synapse to Hijack the Host Endoplasmic Reticulum

Chlamydiae are obligate intracellular bacterial pathogens that replicate within a specialized membrane‐bound compartment, termed an ‘inclusion’. The inclusion membrane is a critical host–pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia‐orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia‐infected cells reveal ‘pathogen synapses’ at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle.     

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus

Membrane traffic between the endoplasmic reticulum and Golgi apparatus is a highly regulated process that uses distinct anterograde and retrograde pathways. These pathways link two organelles that together function as a dynamic membrane system specialized for the biosynthesis and sorting of membrane to be used throughout the cell. The nature and underlying biochemical control of membrane transport along these pathways is thought to be tied to a common regulatory system involving assembly and disassembly of cytosolic proteins on membranes.     

Qualitative and quantitative observations on the structure of the Schwann cells in myelinated fibres

Various morphological features of the Schwann cells of myelinated fibres in the lizard thoracic spinal roots were studied, and, when possible, quantified using morphometric methods. About 0.8% of the Schwann cells are binucleate and some display clusters of microvilli along the internodes. The percentages of the cytoplasmic area of the Schwann cell occupied by the following cytoplasmic components were determined: mitochondria, Golgi apparatus, granular endoplasmic reticulum, smooth endoplasmic reticulum, multivesicular bodies, dense bodies, autophagic vacuoles, peroxisome-like bodies, lipofuscin granules and lipid droplets. Linear relationships were found between the sectional areas of the mitochondria and granular endoplasmic reticulum of the Schwann cell and both the length of the profile of the Schwann cell plasma membrane and the size of the related axon. The results obtained are compatible both with the hypothesis that the mitochondria and granular endoplasmic reticulum of the Schwann cell are involved in the production and storage of proteins for the plasma membrane of this cell, and with the hypothesis that these organelles are involved in the production and storage of protein metabolites which are subsequently transferred to the related axons.     

Drosophila arf72A acts as an essential regulator of endoplasmic reticulum quality control and suppresses autosomal-dominant retinopathy

The eukaryotic endoplasmic reticulum operates multiple quality control mechanisms to ensure that only properly folded proteins are exported to their final destinations via the secretory pathway and those that are not are destroyed via the degradation pathway. However, molecular mechanisms underlying such regulated exportation to these distinct routes are unknown. In this article, we report the role of Drosophila arf72A--the fly homologue of the mammalian Arl1 - in the quality checks of proteins and in the autosomal-dominant retinopathy. ARF72A localizes to the Golgi membranes of Drosophila photoreceptor cells, consistent with mammalian Arl1 localization in cell culture systems. A loss of arf72A function changes the membrane character of the endoplasmic reticulum and shifts the membrane balance between the endoplasmic reticulum and the Golgi complex toward the Golgi complex, resulting in over-proliferated Golgi complexes and accelerated protein secretion. Interestingly, our study indicated that more ARF72A localized on the endoplasmic reticulum in the ninaE(D1) photoreceptor cell, a Drosophila model of autosomal-dominant retinitis pigmentosa, compared to that in the wild-type. In addition, arf72A loss was shown to rescue the ninaE(D1)-related membrane accumulation and the rhodopsin maturation defect, and suppress ninaE(D1)-triggered retinal degeneration, indicating that rhodopsin accumulated in the endoplasmic reticulum bypasses the quality checks. While previous studies of ARF small GTPases have focused on their roles in vesicular budding and transport between the specific organelles, our findings establish an additional function of arf72A in the quality check machinery of the endoplasmic reticulum distinguishing the cargoes for secretion from those for degradation.     

Structural foundations for explaining the physiological roles of murzymes embedded in diverse phospholipid membranes

The advent of improved structural biology protocols and bioinformatics methodologies have provided paradigm-shifting insights on metabolic or physiological processes catalyzed by homo?/hetero- proteins (super)complexes embedded in phospholipid membranes of cells/organelles. In this panoramic review, we succinctly elucidate the structural features of select redox proteins from four systems: hepatocyte/adrenal cortex endoplasmic reticulum (microsomes), inner mitochondrial membrane (cristae), thylakoid membrane (grana), and in the flattened disks of rod/cone cells (in retina). Besides catalyzing fast/crucial (photo)chemical reactions, these proteins utilize the redox-active diatomic gaseous molecule of oxygen, the elixir of aerobic life. Quite contrary to extant perceptions that invoke primarily deterministic affinity-binding or conformation-change based “proton-pump”/“serial electron-relay” type roles, we advocate murzyme functions for the membrane-embedded proteins in these systems. Murzymes are proteins that generate/stabilize/utilize diffusible reactive (oxygen) species (DRS/DROS) based activities. Herein, we present a brief compendium of the recently revealed wealth of structural information and mechanistic concepts on how the membrane proteins use DRS/DROS to aid ‘effective charge separation’ and facilitate trans-membrane dynamics of diverse species in milieu, thereby enabling the cells to function as ‘simple chemical engines’.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

Plant ER geometry and dynamics: biophysical and cytoskeletal control during growth and biotic response

The endoplasmic reticulum (ER) is an intricate and dynamic network of membrane tubules and cisternae. In plant cells, the ER ‘web’ pervades the cortex and endoplasm and is continuous with adjacent cells as it passes through plasmodesmata. It is therefore the largest membranous organelle in plant cells. It performs essential functions including protein and lipid synthesis, and its morphology and movement are linked to cellular function. An emerging trend is that organelles can no longer be seen as discrete membrane-bound compartments, since they can physically interact and ‘communicate’ with one another. The ER may form a connecting central role in this process. This review tackles our current understanding and quantification of ER dynamics and how these change under a variety of biotic and developmental cues.     

Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.     

The widespread occurrence of plant cytosomes resembling animal microbodies

Summary Single membrane bounded organelles characterized by a physical association with endoplasmic reticulum have been observed in a wide range of cell types and plant species including Gymnosperm, Angiosperm, Pteridophyte, and Thallophyte (algae and fungi) tissues. The morphological similarity between these organelles and animal microbodies suggests that they are cytological homologues. Plant microbodies were observed both with and without dense internal inclusions but unlike animal microbodies could not be shown to contain uricase. Plant microbody membranes are resistant to degenerative influences and remain associated with a small portion of endoplasmic reticulum even in isolated cell fractions.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Vps39 is required for ethanolamine-stimulated elevation in mitochondrial phosphatidylethanolamine

Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a ‘moonlighting’ role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ, implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria.     

How the ER stays in shape

Understanding the molecular mechanisms that control the architecture of organelles is an area of intense study. In this issue of Cell, Voeltz et al. (2006) report that two membrane proteins, Rtn4a/NogoA and DP1/Yop1p, are responsible for the generation of tubular morphology in the endoplasmic reticulum (ER). The unusual membrane topology of these proteins may directly contribute to ER curvature.     

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins

Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes.     

Making autophagosomes: Localized synthesis of phosphatidylinositol 3-phosphate holds the clue

Autophagy presents a topological challenge for the cell because it requires delivery of cytosolic material to the lumen of a membrane bound compartment, the lysosome. This is solved in an ingenious way by the formation of a double-membrane vesicle, the autophagosome, which captures cytosolic proteins and organelles during its transformation from a planar membrane disk into a sphere. In this way, cytosolic material first becomes lumenal and is then delivered for degradation to the lysosome. An unsolved set of questions in autophagy concerns the membrane of the autophagosome: what are the signals for its formation and what is its identity? Recently we provided some clues that may help answer these questions.¹ By following the dynamics of several phosphatidylinositol 3-phosphate (PI3P)-binding proteins during amino acid starvation (and autophagy induction) we concluded that at least some autophagosomes are formed in a starvation-induced, PI3P-enriched membrane compartment dynamically connected to the endoplasmic reticulum (ER). We termed the membranes of this compartment omegasomes (from their omega-like shape). Our data suggest that PI3P is important for providing localization clues and perhaps for facilitating the fusion step at the final stage of autophagosome formation.

Addendum to: Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008; 182:685-701.     

Protein bodies of seeds: Ultrastructure,biochemistry, biosynthesis and degradation

Typical organelles for protein storage occur in seeds, protein bodies are found in haploid, diploid or triploid tissues and are single membrane bound. In some plants, they exhibit inclusions (globoid and crystalloid), but not in Gramineae endosperm or in Leguminosae cotyledons. A relationship between species and protein body ultrastructure can be put forward. The chemical composition is based mainly on storage proteins and phytic acid but, hydrolytic enzymes(protease and phytase), cations and ribonucleic acids are also present. Other minor biochemical components include oxalic acid, carbohydrates (excluding starch) and lipids. The locations of the storage proteins, enzymes and phytin are described. Protein body ontogeny during seed maturation has given rise to much controversy: are they plastidic or vacuolar? Recent studies on the location of proteosynthesis show that protein bodies are probably synthesized in endoplasmic reticulum lumen and that the Golgi apparatus plays an important role in storage protein synthesis. During germination protein bodies swell and fuse, giving rise to the cell central vacuole, while the integrity of the membrane is maintained. Protein bodies may be considered as being an example of tonoplast origin from endo-plasmic reticulum.     

Open questions: why should we care about ER-phagy and ER remodelling?

The endoplasmic reticulum (ER) is one of the most complex organelles in the eukaryotic cell. Recent findings suggest that a process called ER-phagy plays a major role in maintaining the ER’s shape and function.