Faculty Opinions recommendation of A network of cytosolic factors targets SRP-independent proteins to the endoplasmic reticulum.

We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca2+ concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.

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Polyubiquitination Is Required for US11-dependent Movement of MHC Class I Heavy Chain from Endoplasmic Reticulum into Cytosol

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2546 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2546-2555 ◽

Cited By ~ 99

Author(s):

Caroline E. Shamu ◽

Dennis Flierman ◽

Hidde L. Ploegh ◽

Tom A. Rapoport ◽

Vincent Chau

Keyword(s):

Endoplasmic Reticulum ◽

Mhc Class I ◽

Heavy Chain ◽

Cell System ◽

Class I ◽

Permeabilized Cell ◽

Heavy Chains ◽

Lysine Residues ◽

Lumenal Domain ◽

Cytosolic Factors

The human cytomegalovirus protein US11 induces the dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol for degradation by the proteasome. With the use of a fractionated, permeabilized cell system, we find that US11 activity is needed only in the cell membranes and that additional cytosolic factors are required for heavy chain dislocation. We identify ubiquitin as one of the required cytosolic factors. Cytosol depleted of ubiquitin does not support heavy chain dislocation from the ER, and activity can be restored by adding back purified ubiquitin. Methylated-ubiquitin or a ubiquitin mutant lacking all lysine residues does not substitute for wild-type ubiquitin, suggesting that polyubiquitination is required for US11-dependent dislocation. We propose a new function for ubiquitin in which polyubiquitination prevents the lumenal domain of the MHC class I heavy chain from moving back into the ER lumen. A similar mechanism may be operating in the dislocation of misfolded proteins from the ER in the cellular quality control pathway.

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Tail-anchored protein biosynthesis at the endoplasmic reticulum: the same but different

Biochemical Society Transactions ◽

10.1042/bst0320659 ◽

2004 ◽

Vol 32 (5) ◽

pp. 659-662 ◽

Cited By ~ 34

Author(s):

S. High ◽

B.M. Abell

Keyword(s):

Endoplasmic Reticulum ◽

Molecular Basis ◽

Protein Biosynthesis ◽

Cytochrome B5 ◽

Membrane Insertion ◽

Essential Step ◽

Protein Biogenesis ◽

Multiple Routes ◽

Membrane Components ◽

Cytosolic Factors

The post-translational integration of tail-anchored proteins at the endoplasmic reticulum represents a novel and distinct pathway for membrane protein synthesis. Studies of various precursors, exemplified by the synaptobrevins and cytochrome b5, indicate that multiple routes may facilitate their biosynthesis. There is clear evidence that both cytosolic factors and membrane components facilitate the efficient membrane insertion of at least some tail-anchored proteins. However, the nature of these mediators is currently unknown and their identification will be an essential step in defining the molecular basis of tail-anchored protein biogenesis.

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Continuous Equilibration of Phosphatidylcholine and Its Precursors between Endoplasmic Reticulum and Mitochondria in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0460 ◽

2003 ◽

Vol 14 (5) ◽

pp. 2142-2150 ◽

Cited By ~ 17

Author(s):

Anton I.P.M. de Kroon ◽

Martijn C. Koorengevel ◽

Tom A.M Vromans ◽

Ben de Kruijff

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Subcellular Fractionation ◽

Unknown Mechanism ◽

Cytosolic Factors

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.

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Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

Molecular Biology of the Cell ◽

10.1091/mbc.9.1.103 ◽

1998 ◽

Vol 9 (1) ◽

pp. 103-115 ◽

Cited By ~ 45

Author(s):

Andrea Neuhof ◽

Melissa M. Rolls ◽

Berit Jungnickel ◽

Kai-Uwe Kalies ◽

Tom A. Rapoport

Keyword(s):

Endoplasmic Reticulum ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Membrane Binding ◽

Membrane Targeting ◽

Signal Recognition ◽

Nascent Polypeptide ◽

Nascent Chain ◽

Cytosolic Factors ◽

Er Membrane

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

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In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050676 ◽

1974 ◽

Vol 32 ◽

pp. 132-133

Author(s):

John J. Wolosewick ◽

John H. D. Bryan

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Endoplasmic Reticulum ◽

Nuclear Ring ◽

Rough Er ◽

Distal Direction ◽

In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

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Ultrastructure of the Parotid Gland of the Nine-Banded Armadillo

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080626 ◽

1977 ◽

Vol 35 ◽

pp. 640-641

Author(s):

J. R. Ruby

Keyword(s):

Endoplasmic Reticulum ◽

Parotid Gland ◽

Periodic Acid ◽

Acinar Cells ◽

Duct System ◽

Parotid Glands ◽

Dasypus Novemcinctus ◽

Anterior Portion ◽

Periodic Acid Schiff ◽

Thin Sections

Parotid glands were obtained from five adult (four male and one female) armadillos (Dasypus novemcinctus) which were perfusion-fixed. The glands were located in a position similar to that of most mammals. They extended interiorly to the anterior portion of the submandibular gland.In the light microscope, it was noted that the acini were relatively small and stained strongly positive with the periodic acid-Schiff (PAS) and alcian blue techniques, confirming the earlier results of Shackleford (1). Based on these qualities and other structural criteria, these cells have been classified as seromucous (2). The duct system was well developed. There were numerous intercalated ducts and intralobular striated ducts. The striated duct cells contained large amounts of PAS-positive substance.Thin sections revealed that the acinar cells were pyramidal in shape and contained a basally placed, slightly flattened nucleus (Fig. 1). The rough endoplasmic reticulum was also at the base of the cell.

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Organisation in the Structure of the Cytoplasmic Ground Substance

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070357 ◽

1978 ◽

Vol 36 (3) ◽

pp. 627-639

Author(s):

K.R. Porter

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Random Distribution ◽

Ground Substance ◽

The Matrix ◽

Cell Processes ◽

Cytoplasmic Ground Substance

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

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