Intracellular pathways of receptor-bound GnRH agonist in pituitary gonadotropes

Summary Localization of GnRH receptors in rat pituitary gonadotropes was studied by use of ¹²⁵I-[azidobenzoyl-D-Lys⁶]GnRH which, upon photolysis, is covalently bound to the receptor molecule. Using high resolution autoradiography, it was found that, after a 90-min incubation of the analog with pituitary cells at 4° C, 93% of the silver grains were associated with the plasma membrane of the gonadotropes. After 45-min incubation of the cells at 37° C, clustering and internalization of the receptor-bound GnRH analog were evident. Silver grains were associated with coated pits, intracellular vesicles, Golgi complexes, lysosome-like structures and secretory granules. The data indicate that receptor-bound GnRH agonist is internalized, at least in part, via coated pits and is subsequently routed to lysosomes where degradation of the hormone-receptor complex may occur. The presence of a considerable amount of silver grains associated with secretory granules may suggest that some of the internalized receptor molecules can escape degradation and be recycled to the cell membrane.     

Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.     

Sequential deposition of plant glycoproteins and polysaccharides at the host-parasite interface ofUromyces vignae andVigna sinensis

Summary Monokaryotic haustoria (M-haustoria) ofUromyces vignae inVigna sinensis cells are surrounded by an extrahaustorial matrix (ema) and the invaginated host plasmalemma, the extrahaustorial membrane (ehrn). The ema was characterized with antibodies against components of the plant cell wall; the ema contained hydroxyproline-rich glycoproteins and arabinogalactans/arabinogalactan proteins, both at a higher concentration close to the ehm. Haustoria with large vacuoles had the ema encased by additional layers. An electron-translucent inner layer deposited on top of the ema contained arabinogalactans/arabinogalactan proteins, hydroxyproline-rich glycoproteins, and callose. The inner layer was surrounded by an electron-translucent middle layer with numerous dark inclusions, rich in pectin and fucose bound to xyloglucans. Finally, a more electron-dense outer layer containing arabinogalactans/arabinogalactan proteins and hydroxyproline-rich glycoproteins encased the whole structure. These polysaccharides, with the exception of callose and un-esterified pectin, were also found in the plant Golgi apparatus. The polysaccharides were synthesized in the trans Golgi cisternae and secreted into the host-parasite interface. The secretory events seem to be coupled to endocytosis since numerous coated pits were found on the ehm too. The pits were elongated, sometimes formed tubules and the coat reacted with an antibody against plant clathrin. Our results suggest intensive membrane recycling around haustoria, together with the secretion of cell wall material, which in the case of more or less vacuolated haustoria seems to be responsible for encasementAbbreviations AG/AGP arabinogalactans and arabinogalactan proteins - BSA bovine serum albumin - ehm extrahaustorial membrane - ema extrahaustorial matrix - HRGP_2b hydroxyproline rich glycoproteins - M-haustorium monokaryotic haustorium - TBS tris buffered saline     

Ultrastructure of Blue-Green Algae

Two freshwater blue-green algae, Tolypothrix tenuis and Fremyella diplosiphon, and an oscillatorialike marine alga, were found to possess structures on the photosynthetic lamellae which appear to correspond to the phycobilisomes of red algae. These homologous structures are important because they contain the phycobilins which are accessory pigments involved in photosynthesis. As in the red algae, the phycobilisomes were attached on the outer side of each lamellae, i.e., the side facing away from its own membrane pair. Although our study on Anacystis nidulans has not thus far revealed the presence of phycobilisomes, some observations were made on the structure of the polyhedral bodies. After negative staining, the polyhedral bodies were seen to be composed of regularly spaced subunits arranged in a crystalline array. Elongated segmented rods, which differed from the polyhedral bodies, were found in the nuclear region of apparently healthy Tolypothrix cells.     

Endocytosis in spermatids during spermiogenesis of the mouse

In the course of spermiogenesis in the mouse, spermatid cytoplasm contains numerous membrane pits, vesicles and membranous tubules which are frequently anastomosed. Pale and dense multivesicular bodies (MVB) and secondary lysosome-like structures are also present in the cytoplasm. In order to study the pathway of non-specific adsorptive endocytosis in spermatids, cationic ferritin (CF) was directly microinjected into the lumen of seminiferous tubules, and added to germinal cell culture. Tissue and cultures were fixed at various time intervals after injection. Two-5 hr after microinjection of tracer, CF was found simultaneously in vesicles, tubules, MVB and in lysosome-like bodies present in spermatids at all steps of spermiogenesis. Various membranous components of the Golgi medulla, and the innermost transsaccule of the Golgi cortex were labelled simultaneously. In primary cultures of spermatids, the vesicles contained the marker 5 min after its deposition; 10 min after deposition, CF was evident in tubules; at 30 min, CF was present in pale MVB; at 1 hr, the dense MVB and lysosome-like bodies were labelled. Finally, at 2 hr 30 min, vesicles and tubules of the Golgi medulla contained CF grains. Apparently spermatids are very active cells in the process of adsorptive endocytosis throughout spermiogenesis. Endocytosis in spermatids is probably one of the mechanisms involved in the uptake of material used to build up spermatozoa components. The strong labelling of the Golgi region probably point to its role in recycling endocytosed membranes.     

Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.     

The biochemical aspects of blood group antigens]

The biochemical aspects of the immunodominant structures of blood groups antigens are mainly restricted to the following: ABH and Lewis in secretory fluids or on the red blood cells; P system (P1, P, Pk antigens); MN antigens and related; Tn and Tn antigens; Some hypothesis may be put forward for the I, i antigens. Many other antigens seem to be on the dependence of interactions between proteins and lipids of the red cell membrane; such immunodominant structures are not yet known. Except for the ABH and Lewis groups, the biosynthesis pathways are at present unclear.     

FINE STRUCTURE IN FROZEN-ETCHED YEAST CELLS

The freeze-etching technique, which is a special kind of freeze-drying, allows electron microscopic investigation of cells and tissues in the frozen state. In regard to yeast cells (Saccharomyces cerevisiae) a freeze-fixation technique has been developed which does not kill the object. The electron micrographs therefore are considered to impart an image of high fidelity. The cutting of the frozen object, which actually consists of a fine splintering, produces not only cross-sectional views (cross-fractures) of the structures but also surface views of the membranes and organelles. Many surface structures are described which have not been shown by the usual sectioning techniques. The cytoplasmic membrane contains hexagonal arrangements of particles which are apparently involved in the production of the glucan fibrils of the cell wall. Alterations of the distribution of nuclear pores are shown in cells of different ages. Freeze-etching enables a clear distinction of endoplasmic reticulum and vacuoles in yeast cells. The membranes of the vesicular systems are covered by ribosomes arranged in circular patterns. The mitochondrial envelope shows small perforations which could allow the exchange of macromolecules. The storage granules consist of concentric layers of lipid, presumably phosphatide. A Golgi apparatus has been detected which may be involved in the storage of lipid. The structure of the unit membrane and the membrane structures of all organelles as revealed by chemical fixation are confirmed in principle. Glycogen agglomerations are identified in the ground plasm of older cells. Insight into artifacts introduced by common chemical fixation and embedding techniques is obtained and discussed.     

On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells

Summary In rabbit luteal cells the transmost element (G₂) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and its was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G₂–G₁) selectively. Controls showed a well-preserved ultrastructure.Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G₂) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity.It is concluded that the trans Golgi network (G₂–G₁) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane streches seem to be involved in endocytosis and in the formation of lysosomal membranes.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.     

Electron microscopical studies on the thyroid of a cyclostome,Lampetra japonica,during its upstream migration

Summary Electron microscopical studies were made of the thyroid gland of an adult lamprey, Lampetra japonica, in the upstream migration period.The thyroid consists of many usual follicles containing the colloid in their lumina, and a large parafollicle without colloid. The paper concerns only the usual follicle.The follicle cells found in the usual follicle wall are classified into three types; 1. a non-ciliated taller cell, 2. a ciliated taller one, and 3. a non-ciliated cuboidal one. From their cytoplasmic fine structure, it is considered that all these cells are essentially identical and differences among them are due to their functional state.All these type cells are characterized by irregularly developed interdigitations and aggregates of tonofilaments throughout the cytoplasm, especially in the perinuclear region. Although the rough-surfaced endoplasmic reticulum and the Golgi apparatus are fairly well developed in the first and second type cells, the cisternae are not so large-vacuolated but flattened, and the cytoplasm is more compact as compared with that of the higher vertebrate. In the third type cell, the cytomembranes are poorly developed.Large dense inclusion-bodies consisting of heterogeneously dense materials, of lamellar structures, and of less dense vacuoles, which are found often in taller follicle cells, are also characteristic for the lamprey thyroid. The body which might be intimately related to the Golgi apparatus is considered to be a kind of lysosomes and it perhaps corresponds to the yellow pigment observed by light microscopy.In the apical part of the cytoplasm in taller cells, there are three kinds of granules or vesicles; numerous small vesicles considered to be derived from the Golgi apparatus, a few small dense granules which seem to originate from the Golgi region, and a few large less-dense granules.In the third type cell, the cytomembranes are not so well developed as those of the first and second type cells. The large heterogeneously dense bodies and the cytoplasmic granules are very few in number.Around the follicle of the lamprey thyroid, there are a dense basement membrane and a relatively compact connective tissue with few blood capillaries. Characteristic fat cells are found in the connective tissue.     

On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells

In rabbit luteal cells the transmost element (G2) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and it was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G2-G1) selectively. Controls showed a well-preserved ultrastructure. Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G2) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity. It is concluded that the trans Golgi network (G2-G1) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane stretches seem to be involved in endocytosis and in the formation of lysosomal membranes.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

Cytological studies of developing muscle with special reference to myofibrils,mitochondria, Golgi material and nuclei

Summary Developing striated muscle in the chick embryo was studied with special reference to the cytological changes which occur as the myofibrils are being elaborated. Observations were made on the mitochondria, cytoplasmic granules, Golgi material, neutral red bodies, nuclei, and nucleoli, and on the developing fibrils. Particular attention was directed to the early part of the histogenetic process when a few primary myofibrils are being formed and becoming striated. This period is characterized by hypertrophy of the Golgi material, nuclei, and nucleoli.The myofibrils were found to arise in close association with filamentous mitochondria, apparently at the expense of the numerous small cytoplasmic granules which fill the early myoblasts. The mitochondria change staining reaction during this time and to a certain extent disappear. Many ring-shaped mitochondria, the significance of which is not known, were found among the unchanged mitochondria which remain near the nucleus.At first the myofibrils are homogeneous and do not take the stain well. Later they stain heavily, although they remain homogeneous, and finally striation appears. Evidence was presented that the Z-membrane begins development before striation is visible.When the primary fibrils have become striated, formation of myofibrils by the above method apparently ceases and further increase in the number of fibrils is probably brought about by longitudinal splitting of those formed first.At this time, also, the Golgi material decreases in amount, the nuclei become smaller, and the nucleoli break up and lose their distinct form.A secondary fibril system was also described which, it is believed, is distinct from the myofibril system.Mitotic division followed by cleavage of the cytoplasm occurs in the earliest myoblasts, probably for the purpose of increasing the number of these cells. However, as soon as fibrillization sets in, mitotic division ceases and the nuclei multiply by amitosis not followed by cleavage of the cytoplasm.The nucleoli divide before amitotic division of the nuclei. Evidence was presented that certain nuclear granules may be associated with the amitotic division process.The Golgi material in embryonic muscle is situated at the two poles of the nuclei. This furnishes evidence in favor of the belief that the Golgi material of adult muscle has a similar location and is not represented by the Cajal-Fusari network.Neutral red granules were demonstrated near the nucleus and in the axial cytoplasm, but they did not coincide in position with the Golgi material.It was noted that the cytological changes which take place when the myofibrils are being elaborated in the developing myoblasts resemble those in gland cells at the time secretory materials are being formed.     

大豆根瘤侵染细胞中一种细胞质内含物的电镜观察

在中国丰收11号大豆根瘤侵染细胞中,我们发现了一种电子密度很高,体积很大,形状为圆形或近似圆形,外面没有界膜,常位于胞间隙附近的特殊的细胞质内含物。高尔基体及其小泡,丰富的粗糙型内质网和核糖体常在它的附近,其中一些核糖体正沉积在它的表面。它主要是由核糖体凝聚而成,高尔基体和内质网在它的形成中也起了一定作用。它的内部含有颗粒状,纤维状,泡状和管状物质。它的出现似乎与侵染细胞固氮有关。     

Further studies of the glandular tissue of the Sauromatum guttatum (Araceae) appendix

Electron microscopic studies showed that the trans-Golgi network (trans indicates the polarity of cisternae within the Golgi apparatus; it is opposite to the cis-face that is adjacent to the rough endoplasmic reticulum) was involved in the processing of the osmiophilic material present in the appendix of the inflorescence of Sauromatum guttatum. This material accumulated in the rough endoplasmic reticulum and in special pockets of the plasma membrane prior to heat production. Associations between the endoplasmic reticulum and trans-Golgi network were observed. The Golgi apparatus was composed of 5–6 dictyosomes on one side and one or two somewhat detached cisternae on the other side. Various nonosmiophilic Golgi-derived vesicles were observed: small ones covered with spike-like material, large ones with a smooth surface, and irregularly shaped ones. These electron-translucent vesicles seemed to accumulate in specific localities at the plasma membrane surface in the vicinity of the osmiophilic material; they were not found when the aroma was released. During heat production, the Golgi structures shrank and the activity of the trans-Golgi network seemed to be reduced. At the same time, coated pits were seen at the plasma membrane surface. In some cells, hypertrophic Golgi apparatuses were seen with only 2–3 dictyosomes that contained granulated material in their lumens. Finally, the osmiophilic material was also found in the plasmodesmata.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM RUBRUM

Sections of Rhodospirillum rubrum cells from cultures of different ages have been examined to obtain information on the development of chromatophores in this organism. Cells from the 12-hour cultures studied contain neither distinct invaginations of the cytoplasmic membrane nor distinct chromatophores. The first structures that can be related to chromatophore development occur peripherally in the cells, are relatively few in number, relatively high in density, and have an indistinct membrane. In cells from 26-hour cultures numerous distinct invaginations of the cytoplasmic membrane are present, and all layers of the cytoplasmic membrane are involved in the formation of each invagination. As the invaginations become more numerous, the ends of the invaginations become constricted to form one or more structures similar to the chromatophores previously described in this organism. Cells of R. rubrum, therefore, develop a structural continuum which initially consists of invaginations of the cytoplasmic membrane, and later of the chromatophores produced by and attached to these invaginations. The presence of this continuum, however, does not necessarily exclude the existence of discrete chromatophores within these cells. Several other structures previously reported in this organism are described in greater detail.     

Assembly of plasmid DNA and chromatophore inRhodospirillum rubrum

Summary Rhodospirillum rubrum, a photosynthetic bacterium, contains many photosynthetic vesicular membranous structures called chromatophores. The organism contains a 55 kb specific plasmid which is essential for photosynthesis, but the exact relationship between the chromatophore and the plasmid is uncertain. In this study we examined the precise localization of the plasmids, especially in relation to the chromatophores. Fluorescence in situ hybridization indicated that there are several copies of the plasmid per cell and that some plasmids are localized close to the cellular envelope. In situ hybridization at the electron-microscopic level further revealed that the plasmid localized to the periphery of the chromatophore close to the envelope. Moreover, when the chromatophore fraction was purified from cells, the plasmid DNA was observed as a cluster around the chromatophore vesicles. The assembly of the plasmid and chromatophore may be related to chromatophore formation by invagination of cell membrane.     

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.