Actin-endoplasmic reticulum complexes inDrosera

In epidermal cells ofDrosera tentacles that have been preserved for ultrastructural analysis through high pressure freeze fixation and freeze substitution we describe the frequent occurrence of microfilament (MF)-endoplasmic reticulum (ER) complexes. These are found throughout the cytoplasm where they are observed in close association with the plasmalemma (PL), the tonoplast, nuclei, mitochondria, chloroplasts, and microbodies. The MF component of the complexes is identified as actin based on immunogold labelling with actin antibodies. The actin-ER complexes are prominent in the cortical cytoplasm. In this region a network of predominantly tubular ER occupies an intermediary position in which it associates closely with both the PL and the actin MFs. We suggest that the ER, especially those elements adjacent to the PL in the cortical cytoplasm, stabilizes the actin MFs and provides the necessary anchor against which the forces for cytoplasmic streaming are generated.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FS freeze substitution - HPF high pressure freezing - MF microfilaments - MT microtubules - PL plasmalemma     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.Abbreviations EGTA ethylene glycol bis-(-aminoethyl ether), N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - MF microfilament - MT microtubule - PEG polyethylene glycol - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution     

Intrinsically Disordered Linker and Plasma Membrane‐Binding Motif Sort Ist2 and Ssy1 to Junctions

Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)‐resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM‐binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.      

Organelles involved in the synthesis and transport of hydroxyproline-containing glycoproteins in carrot root discs

Isopycnic sucrose density gradients of homogenates from carrot root tissue were analyzed radiochromatographically, radiochemically, and photometrically for the presence of hydroxyproline residues. Significant amounts were found in endoplasmic reticulum (ER), Golgi apparatus (GA) and plasma membrane (PM) fractions as designated by the presence of marker enzymes for these membranes. Some hydroxyproline-containing macromolecules could be detected in the soluble cytoplasm (cytosol) but this was interpreted as an artefact due to homogenization. Hydroxyproline-rich polymers can be released from a mixed ER/PM fraction by freezing and thawing in water. The PM-associated hydroxyproline polymer is suggested to be an arabinogalactan protein rather than cell wall extensin. Nevertheless, the polypeptides of both glycoproteins are considered as being synthesized at the ER and transported via the GA to the PM.Abbreviations BSA Bovine serum albumin - CCO cytochrome c-oxidase - CCR cytochrome c-reductase - DTT dithiothreitol - EDTA ethylene diaminotetracetic acid - ER endoplasmic reticulum - GA Golgi apparatus - GS I/II glucan synthetase I/II - IDP(ase) inosine diphosphat(ase) - PM plasma membrane - RNA ribonucleic acid - TCA trichloracetic acid - Tris tris-(hydroxymethyl)-aminomethane - UDPG uridine diphosphoglucose     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Tension in tubulovesicular networks of Golgi and endoplasmic reticulum membranes

The endoplasmic reticulum (ER) and Golgi have robust bidirectional traffic between them and yet form distinct membrane compartments. Membrane tubules are pulled from large aggregates of ER or Golgi by microtubule motors to form ER tubulovesicular networks or Golgi tubules both in vivo and in vitro. The physical properties of membranes are critical for membrane traffic and organelle morphology. For example, tension applied to membranes can create tethers, drive membrane flow, and set the diameter of the tubules. Here, we formed ER and Golgi membrane networks in vitro and used optical tweezers to measure directly, for the first time, the membrane tensions of these organelles to clarify the possible role of tension in membrane flow. We report that higher forces are needed to form tethers from ER (18.6 +/- 2.8 pN) than from Golgi (11.4 +/- 1.4 pN) membrane tubules in vitro. Since ER tubules are smaller in diameter than Golgi tubules, it follows that Golgi networks have a lower tension than ER. The lower tension of the ER could be an explanation of how Golgi tubules can be rapidly drawn into the ER by tension-driven flow after fusion, as is observed in vivo.     

Morphological and functional aspects of STIM1-dependent assembly and disassembly of store-operated calcium entry complexes

The SOCE (store-operated Ca2+ entry) pathway is a central component of cell signalling that links the Ca2+-filling state of the ER (endoplasmic reticulum) to the activation of Ca2+-permeable channels at the PM (plasma membrane). SOCE channels maintain a high free Ca2+ concentration within the ER lumen required for the proper processing and folding of proteins, and fuel the long-term cellular Ca2+ signals that drive gene expression in immune cells. SOCE is initiated by the oligomerization on the membrane of the ER of STIMs (stromal interaction molecules) whose luminal EF-hand domain switches from globular to an extended conformation as soon as the free Ca2+ concentration within the ER lumen ([Ca2+]ER) decreases below basal levels of ~500 μM. The conformational changes induced by the unbinding of Ca2+ from the STIM1 luminal domain promote the formation of higher-order STIM1 oligomers that move towards the PM and exposes activating domains in STIM1 cytosolic tail that bind to Ca2+ channels of the Orai family at the PM and induce their activation. Both SOCE and STIM1 oligomerization are reversible events, but whether restoring normal [Ca2+]ER levels is sufficient to initiate the deoligomerization of STIM1 and to control the termination of SOCE is not known. The translocation of STIM1 towards the PM involves the formation of specialized compartments derived from the ER that we have characterized at the ultrastructural level and termed the pre-cortical ER, the cortical ER and the thin cortical ER. Pre-cortical ER structures are thin ER tubules enriched in STIM1 extending along microtubules and located deep inside cells. The cortical ER is located in the cell periphery in very close proximity (8-11?nm) to the plasma membrane. The thin cortical ER consists of thinner sections of the cortical ER enriched in STIM1 and devoid of chaperones that appear to be specialized ER compartments dedicated to Ca2+ signalling.     

A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

We analyzed the structure of yeast endoplasmic reticulum (ER) during six sequential stages of budding by electron tomography to reveal a three-dimensional portrait of ER organization during inheritance at a nanometer resolution. We have determined the distribution, dimensions, and ribosome densities of structurally distinct but continuous ER domains during multiple stages of budding with and without the tubule-shaping proteins, reticulons (Rtns) and Yop1. In wild-type cells, the peripheral ER contains cytoplasmic cisternae, many tubules, and a large plasma membrane (PM)-associated ER domain that consists of both tubules and fenestrated cisternae. In the absence of Rtn/Yop1, all three domains lose membrane curvature, ER ribosome density changes, and the amount of PM-associated ER increases dramatically. Deletion of Rtns/Yop1 does not, however, prevent bloated ER tubules from being pulled from the mother cisterna into the bud and strongly suggests that Rtns/Yop1 stabilize/maintain rather than generate membrane curvature at all peripheral ER domains in yeast.     

Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Store-operated Ca2+ entry: evidence for a secretion-like coupling model.

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.     

Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces.     

Five Arabidopsis Reticulon Isoforms Share Endoplasmic Reticulum Location,Topology, and Membrane-Shaping Properties

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.     

Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

A Highlights from MBoC Selection: Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.     

Increase in the number of contacts of endoplasmic reticulum with mitochondria and plasma membrane in yeast cells stimulated to division with He-Ne laser light

We studied the ultrastructure of Torulopsis sphaerica yeast cells irradiated with He-Ne laser (lambda = 632.8 nm, dose--460 J/m2) and then cultured for 6 h in the nutrient with 1% glucose by aeration. The length of membranes of endoplasmic reticulum (ER) and the number of its associations with mitochondria (M) and plasma membrane (PM) were measured on ultrathin sections. A distance of less than 50 nm between heterogeneous membranes was considered as an "association". The cells from irradiated cultures are characterized by the following features: 1) the length of cortical ER membranes in relation to cellular perimeter, and the length of perinuclear ER membranes in relation to nuclear perimeter increase, resp., by 21 and 79%; 2) the number of ER-PM associations per cellular section, and that per unit of PM length increase, resp., by 26 and 41%; 3) the number of ER-M association in relation to the total mitochondrial perimeter, and to perimitochondrial ER increase by 80 and 87%, resp. The latter may be associated with Ca2+ uptake by mitochondria associated with ER, which results in activation of respiration and ATP production.     

Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen with green fluorescent protein

Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.     

Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.     

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.