scholarly journals Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

1998 ◽  
Vol 140 (3) ◽  
pp. 553-563 ◽  
Author(s):  
Alicia Llorente ◽  
Andrzej Rapak ◽  
Sandra L. Schmid ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Trichostatin A ◽  
Selective Inhibition ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tyrosine Sulfation ◽  
Cell Biol ◽  
The Difference ◽  
In Cells

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

scholarly journals Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface.

1988 ◽  
Vol 107 (5) ◽  
pp. 1655-1667 ◽  
Author(s):  
E Friederich ◽  
H J Fritz ◽  
W B Huttner
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
Secretory Protein ◽  
Reversible Inhibitor ◽  
Secreted Protein ◽  
Half Time ◽  
Wild Type ◽  
Tyrosine Sulfation ◽  
L Cell ◽  
Cell Clones

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals Defect in the p53-Mdm2 Autoregulatory Loop Resulting from Inactivation of TAFII250 in Cell Cycle Mutant tsBN462 Cells

2000 ◽  
Vol 20 (15) ◽  
pp. 5554-5570 ◽  
Author(s):  
Christine Wasylyk ◽  
Bohdan Wasylyk
Keyword(s):  
Cell Cycle ◽  
Temperature Sensitive ◽  
Gene Promoters ◽  
Wild Type ◽  
Promoter Activation ◽  
Cycle Arrest ◽  
Mdm2 Expression ◽  
Temperature Sensitive Phenotype ◽  
Stress Activation ◽  
In Cells

ABSTRACT The cell cycle arrest and proapoptotic functions of p53 are under tight control by Mdm2. After stress activation of p53 by nontranscriptional mechanisms, transcription of the mdm2gene results in increased synthesis of Mdm2 and down-regulation of p53. Disruption of this autoregulatory loop has profound effects on cell survival and tumorigenesis. We show that a defective p53-Mdm2 autoregulatory loop results from inactivation of a basal transcription factor, TAFII250, in tsBN462 cells. We found that Mdm2 expression rescues the temperature-sensitive phenotype of tsBN462 cells, as shown by activation of cell cycle-regulated gene promoters (B-myb, cyclin A, and cdc25C), increased cell growth and DNA synthesis, and inhibition of apoptosis. These effects of Mdm2 are mediated by p53. Exogenous Mdm2 expression apparently complements endogenous Mdm2 synthesis in tsBN462 cells, which is reduced compared to that in the equivalent parental cells with wild-type TAFII250, BHK21. Expression of wild-type TAFII250 in tsBN462 stimulates and prolongs the synthesis of Mdm2 and rescues the temperature-sensitive phenotype. The TAFII250 rescue is blocked by inhibition of Mdm2-p53 interactions. We also show that Mdm2 promoter activation, after transfer to the nonpermissive temperature, is attenuated in cells with mutant TAFII250. The temperature-sensitive phenotype apparently results from inefficient inhibition of heat-induced p53 by reduced Mdm2 synthesis due to low mdm2 promoter activity. These results raise the possibility that the p53-Mdm2 autoregulatory loop could guard against transcriptional defects in cells.

scholarly journals Inhibition by cyanate of the processing of lysosomal enzymes

1983 ◽  
Vol 210 (3) ◽  
pp. 795-802 ◽  
Author(s):  
A Hasilik ◽  
R Pohlmann ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Cultured Human Fibroblasts ◽  
High Mannose ◽  
In Cells

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.

Studies on the levels of cyclic AMP in cells transformed by wild-type and temperature-sensitive Kirsten sarcoma virus

Cell ◽  
1974 ◽  
Vol 1 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Richard A. Carchman ◽  
George S. Johnson ◽  
Ira Pastan ◽  
Edward M. Scolnick
Keyword(s):  
Cyclic Amp ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Sarcoma Virus ◽  
In Cells

scholarly journals Variants of vaccinia virus hemagglutinin altered in intracellular transport.

1986 ◽  
Vol 6 (11) ◽  
pp. 3734-3745 ◽  
Author(s):  
H Shida
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Slow Movement ◽  
Wild Type ◽  
Sequence Analyses ◽  
In The Wild ◽  
Virus Mutant

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

scholarly journals BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast.

1995 ◽  
Vol 130 (1) ◽  
pp. 41-49 ◽  
Author(s):  
J F Simons ◽  
S Ferro-Novick ◽  
M D Rose ◽  
A Helenius
Keyword(s):  
Molecular Chaperone ◽  
Intracellular Transport ◽  
Critical Role ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Yeast Strains ◽  
Mutant Strains

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER-retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.

Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic

2001 ◽  
Vol 114 (20) ◽  
pp. 3685-3694
Author(s):  
Thomas K. Graves ◽  
Shilpa Patel ◽  
Priscilla S. Dannies ◽  
Patricia M. Hinkle
Keyword(s):  
Growth Hormone ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Unfolded Protein Response ◽  
Wild Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Trh Receptor ◽  
In Cells

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Δ32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Δ32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Δ32-71 growth hormone were dually stained for growth hormone and the Golgi markers β-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but β-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Δ32-71 growth hormone. Examination of α-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Δ32-71 growth hormone. To determine whether Δ32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Δ32-71 growth hormone. Cells expressing Δ32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Δ32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Δ32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

scholarly journals Associations of elements of the Golgi apparatus with microtubules.

1984 ◽  
Vol 99 (3) ◽  
pp. 1092-1100 ◽  
Author(s):  
A A Rogalski ◽  
S J Singer
Keyword(s):  
Golgi Apparatus ◽  
Cultured Cells ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Free Microtubules ◽  
Vesicular Stomatitis ◽  
In Cells

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.

Sign in / Sign up

Export Citation Format

Share Document