Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

Shuttle-like movements of Golgi vesicles in the tip of growingChara rhizoids

Summary In tip-growingChara rhizoids, the in-vivo saltatory movements of Golgi vesicles were recorded. The movements in radial direction back and forth between the ER aggregate and the plasma membrane occurred three times more often than movements passing the ER aggregate tangentially. The mean velocity of the class of Golgi vesicles observed (0.4–1 m in diameter) was approx. 0.3 m/s. Higher speed of 1–1.5 m/s occurred only in radial directions. Possibly, the ER aggregate is involved in guidance of the Golgi vesicles.Abbreviations DIC differential interference contrast - ER endoplasmic reticulum - OsFeCN osmium tetroxide-potassium ferricyanide Dedicated to the memory of Professor O. Kiermayer     

Satnav for cells: Destination membrane fusion

All eukaryotic cells, from budding yeast to plants and mammals, are elaborately subdivided into functionally distinct, membrane-enclosed compartments – or organelles. Each organelle contains its own characteristic set of enzymes and other specialized molecules, which allows for the segregation of distinct biochemical reactions. A complex distribution system transports specific products (or cargos) from one compartment to another, involving a cycle of trafficking vesicle formation from a precursor membrane, vesicle transport to its destination (which may involve use of the cytoskeleton and specific motor proteins) and finally vesicle fusion with its target membrane.In the central nervous system (CNS), rapid communication between neurons at synapses is achieved using such a specialized trafficking pathway. Small synaptic vesicles move to the presynaptic plasma membrane where they fuse in response to Ca²⁺ influx, releasing chemical messengers (neurotransmitters) into the synaptic cleft. Vesicles are then recovered, reformed and refilled with neurotransmitter, ready for subsequent rounds of release. This recycling process may involve fusion with, and reformation from, a specific endosomal recycling station.As correct recycling of synaptic vesicles is essential to maintain neuronal signaling, every aspect of the process has been intensively studied. Amazingly, the general principals elucidated in this system are shared across membrane trafficking pathways in eukaryotes, and are largely mediated by common protein-based machineries. Hence, in this article, I will use the example of neuronal exocytosis to illustrate concepts which currently dominate our thinking about membrane trafficking pathways. In particular, I intend to focus on the all-important issue of how specificity in vesicle transport and fusion is achieved.

1. Download : Download high-res image (136KB)
2. Download : Download full-size image

     

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

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.     

Membrane traffic from the endoplasmic reticulum to the Golgi apparatus is disturbed by an inhibitor of the Ca2+-ATPase in the ER

Summary An electron microscopic study of cress (Lepidium sativum L.) roots treated with cyclopiazonic acid (CPA), an inhibitor of the Ca²⁺-ATPase in the endoplasmic reticulum (ER) has been carried out. Drastic changes in the endomembrane system of the secretory root cap cells were observed. After treatment with CPA dense spherical or elliptoidal aggregates of ER (diameter 2–4 m) were formed in addition to the randomly distributed ER cisternae characteristic for control cells. The formation of ER aggregates indicates that in spite of an inhibition of the Ca²⁺ -ATPase in the ER by CPA, membrane synthesis in the ER continued. The ER aggregates are interpreted as a reservoir of ER membrane material newly synthesized during the 2 h CPA-treatment. Hypertrophied Golgi cisternae and secretory vesicles, which are characteristic for secretory cells under control conditions, were completely absent. Additionally the shape of the Golgi stacks was flat and the diameter of the cisternae was shortened by about one third. These phenomena are indicative of an inactive state of the Golgi apparatus. The cellular organization of both other cell types of the root cap, meristematic cells and statocytes, was not visibly affected by CPA, both having a relatively low secretory activity. The formation of ER aggregates as well as the reduction of Golgi compartments are indications for the existence of a unidirectional transport of membrane material from the ER to the Golgi. It is suggested that the membrane traffic from the ER to the Golgi apparatus is regulated by the cytosolic and/or luminal calcium concentration in secretory cells of the root cap.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum     

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

Summary Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the dark vesicles (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.     

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Summary Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain Golgi equivalents — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 g/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 g/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.Abbreviations BFA brefeldin A - ConA concanavalin A - ER endoplasmic reticulum - PDA potato dextrose agar - RER rough endoplasmic reticulum     

Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10–15 m) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.     

The tanycyte of the rat median eminence

Summary The present ultrastructural study proves the existence of nerve terminals closely apposed to the plasmalemmata of tanycytes in the rat median eminence. Several of these axo-tanycytic endings display remarkable accumulations of agranular endoplasmic reticulum in the form of pleomorphic vesicles which are closely apposed on either side of the plasma membrane of each cell compartment. Some of these vesicular profiles give the impression of structural continuity across both membrane systems. This phenomenon is discussed in the context of being a potential substratum for communication between both cell compartments.Supported by NIH Grant NS13717 and NSF Grant BNS 78-11820     

Shrinkage and fragmentation of the trans-Golgi network in non-meristematic plant cells

Golgi products are exported from the trans-Golgi network (TGN) where they are sorted and packaged into secretory and clathrin-coated vesicles. We have examined TGN cisternae in Arabidopsis root columella cells and in maize basal endosperm transfer cells by electron microscopy/tomography. In these cell types, sizes of the TGN compartments decrease as they produce vesicles. After released from the Golgi, free TGN compartments continue to contract and they were seen to fragment into clusters of vesicles. The shrinkage of the plant TGN and its final disassembly suggest that the plant TGN is not a long-lasting organelle that is replenished regularly by membrane trafficking.Key words: trans-Golgi network, Golgi stack, root columella cell, basal endosperm transfer cell, secretory vesicle, clathrin-coated vesicle, electron tomographyThe TGN refers to a membranous compartment located on the trans-side of the Golgi stack, which sorts Golgi products according to their final destinations.¹ In plant cells, in which Golgi stacks are discrete and mobile, a trans-most Golgi cisterna transforms into a TGN cisterna and the TGN cisterna, later, peels away from the Golgi.² Once separated, movements of the Golgi and of the free TGN compartment are not coupled.^3,4Arabidopsis meristematic cells are small, averaging about 204 µm³ in volume.⁵ Golgi mobility is more restricted in small meristematic cells than in large vacuolated cells such as tobacco BY2 cells.^6,7 In these smaller cells, multiple TGN cisternae often remain associated with their original Golgi stacks, facilitating examination of the emergence of a TGN compartment and its subsequent maturation. We took advantage of the spatial proximity in Arabidopsis meristematic cells to delineate morphological features and protein localizations in the Golgi-associated (GA-) TGN and in free TGN.⁸ Our major findings include:(1) Transformation of a trans-Golgi cisterna into a GA-TGN cisterna involves the formation of secretory vesicle (SV) buds in the outer rim of the cisterna and a 30–35% reduction in cisternal membrane area.(2) RabA4b and phospatidylinositol-4-kinase β1 are associated with the GA-TGN and with the free TGN compartments, but are not associated with trans-Golgi cisternae.(3) Free TGN compartments fragment into SVs and clathrin-coated vesicles (CCVs) and into residual membrane pieces.In this addendum, electron microscopy/tomography analyses of the TGN in two non-meristematic cell types, namely Arabidopsis gravity-sensing root columella cells and maize basal endosperm transfer cells (BETCs), are reported. Formation and maturation of the TGN in these cell types agree with our findings from the meristematic TGN. Free TGN compartments are more abundant in these cell types than in the meristematic cells, facilitating examination of free TGN compartments. Withering and fragmentation of the free TGN compartments in these cell types suggest that the TGN is not a persistent organelle like the Golgi apparatus, which regularly revisits ER export sites to be sustained by the COPII vesicular transport system.     

Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.     

ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix

Plant Golgi stacks are mobile organelles that can travel along actin filaments. How COPII (coat complex II) vesicles are transferred from endoplasmic reticulum (ER) export sites to the moving Golgi stacks is not understood. We have examined COPII vesicle transfer in high-pressure frozen/freeze-substituted plant cells by electron tomography. Formation of each COPII vesicle is accompanied by the assembly of a ribosome-excluding scaffold layer that extends approximately 40 nm beyond the COPII coat. These COPII scaffolds can attach to the cis-side of the Golgi matrix, and the COPII vesicles are then transferred to the Golgi together with their scaffolds. When Atp115-GFP, a green fluorescent protein (GFP) fusion protein of an Arabidopsis thaliana homolog of the COPII vesicle-tethering factor p115, was expressed, the GFP localized to the COPII scaffold and to the cis-side of the Golgi matrix. Time-lapse imaging of Golgi stacks in live root meristem cells demonstrated that the Golgi stacks alternate between phases of fast, linear, saltatory movements (0.9-1.25 microm/s) and slower, wiggling motions (<0.4 microm/s). In root meristem cells, approximately 70% of the Golgi stacks were connected to an ER export site via a COPII scaffold, and these stacks possessed threefold more COPII vesicles than the Golgi not associated with the ER; in columella cells, only 15% of Golgi stacks were located in the vicinity of the ER. We postulate that the COPII scaffold first binds to and then fuses with the cis-side of the Golgi matrix, transferring its enclosed COPII vesicle to the cis-Golgi.     

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 m and is positioned in the center of the apical Golgi-vesicle accumulation (Spitzenkörper). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.Abbreviations CB cytochalasin B - DIC differential interference contrast microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum     

Analysis of COPII Vesicles Indicates a Role for the Emp47–Ssp120 Complex in Transport of Cell Surface Glycoproteins

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium‐binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47–Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.      

Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.     

Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack

An N-ethylmaleimide-sensitive transport component (NSF) has been purified on the basis of its ability to support transport between Golgi cisternae. We now report that NSF is needed for membrane fusion. Thus, when NSF is withheld from incubations of Golgi stacks with cytosol and ATP, uncoated transport vesicles accumulate. Biochemical experiments confirm this conclusion and reveal that NSF is needed to form the first of two previously described prefusion complexes. NSF, therefore, acts within a cascade in which a vesicle-cisterna complex is matured until it is competent for fusion. We suggest that this reflects the stepwise assembly of a multisubunit "fusion machine" following vesicle attachment.     

Development and ultrastructure of the marine,parasitic Oomycete,Lagenisma coscinodisci Drebes (Lagenidiales): Formation of the primary zoospores and their release

Summary Zoosporogenesis inLagenisma begins after the final nuclear division by the development of encystment vesicles which presumably are derived from Golgi vesicles. The sporangial wall is secreted simultaneously. Initially, the encystment vesicles have an internal coat of fine ribs which becomes a uniform mass during the complicated invagination of the vesicles. When the sporangial wall is complete the protoplast cleaves centripetally by means of narrow cleavage cisternae apparently coming from the distal face of the dictyosomes and being detached by interposing ER cisternae. The cleavage cisternae fuse with each other and with the plasmalemma to which they are often parallel. Narrow cytoplasmic compartments are then cut off and swell to become separation vesicles which lie between the developing zoospores but later disintegrate. Basal bodies develop from procentrioles after the final nuclear division and elongate into flagella (without participation of a flagellar vesicle) when cleavage is complete. The mastigonemes are formed within the ER, mature within the peripheral elements of the dictyosomes near the flagellar bases and appear to be extruded after the elongation of the flagellum. Structurally, especially in the organization of the flagellar root apparatus, the zoospores resemble primary zoospores of other Oomycetes. They become motile within the zoosporangium but seem to be driven out by means of additional unknown forces.—Formation of the encystment vesicles and the manner of cleavage are compared with those of other Oomycetes and general aspects ofLagenisma zoosporogenesis are discussed.     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

Sequential intermediates in the transport of protein between the endoplasmic reticulum and the Golgi

Semi-intact cells, a cell population in which the plasma membrane is perforated to expose intact intracellular organelles (Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534), efficiently reconstitute vesicular trafficking of protein from the endoplasmic reticulum (ER) to the cis Golgi compartment. We now extend these studies to biochemically dissect transport of protein between the ER and the Golgi into a series of sequential intermediate steps involved in the budding and fusion of carrier vesicles. At least two broad categories of transport intermediates can be detected, those that involve early steps in transport and those involved in late, fusion-related events. Early transport steps require the transport of protein through a novel intermediate compartment in which protein accumulates at reduced temperature (15 degrees C). We demonstrate that both entry and exit from this 15 degrees C compartment can be successfully reconstituted in vitro. A late step in delivery of protein to the cis Golgi compartment requires Ca2+ (pCa7) and is coincident with a step which is sensitive to a peptide analog which blocks interaction between the Rab family of small GTP-binding proteins and a downstream effector protein(s) (Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2384). The combined results suggest that a single round of vesicular transport between the ER and the Golgi involves a rapid transit through N-ethylmaleimide-sensitive, guanosine 5'-(3-O-thio)triphosphate-sensitive, ATP- and cytosol-dependent step(s) involved in vesicle formation or transport to a novel intermediate compartment, followed by a regulated fusion event triggered in the presence of Ca2+ and functional components interacting with member(s) of the Rab gene family.