The effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane (original) (raw)
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Journal of Cell Biology, 1984
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 [3SS]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.
Journal of Cell Biology, 1998
Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport mac...
Experimental Cell Research, 1996
cretory cell lines show that clusters of small vesicles, Vero cells were infected with the ts-045 strain of ve-short tubules, and pleomorphic structures, designated sicular stomatitis virus, and the cells were incubated IC 2 or ERGIC [7-9; the term IC will be used throughat 39ЊC to accumulate the mutant G glycoprotein in out this paper], are involved in the anterograde export the ER as a misfolded aggregate. Cycloheximide was pathway (ER to Golgi complex). However, it is still deadded to the culture medium 3.5 h after infection to bated whether these structures represent a new comprevent further protein synthesis, and the temperapartment or whether they are a subregion of either the ture was lowered to 10, 15, or 31ЊC. At these tempera-ER or the Golgi complex. The IC could be related to the tures, the mutant G glycoprotein correctly folds and classical transitional elements described in specialized oligomerizes. Immunofluorescence light microscopy secretory cells [2, 10]; however, there is evidence that showed that the G glycoprotein was exported to the the IC is part of the forming, cis-face of the Golgi com-Golgi complex at 31ЊC and to the intermediate complex, hence the term cis-Golgi network [11]. On the partment (IC) at 15ЊC, but no export was observed at other hand, several studies suggest that IC structures 10ЊC. However, incubations at 10ЊC followed by shift are connected by convoluted thin tubules to the ER, to 15 or 31ЊC resulted in the normal transfer of the and represent the exit sites where transport vesicles glycoprotein to the IC and the Golgi, respectively. Imdestined for the Golgi complex are formed 12]. Other munoelectron microscopical analysis confirmed all data indicate that the IC is not in physical contact with these results, but showed also that the glycoprotein either the ER and or the Golgi complex and is present was frequently clustered in the ER at 10ЊC. Conveneven very far from the Golgi stacks 13, 14]. Results tional electron microscopy showed that the morpholobtained with a cell-free system support the existence ogy of the ER, IC, and Golgi complex remained essenof an intermediate step in the anterograde transport tially unchanged at all temperatures. The only signifi-[15, 16]. No enzymatic activities have yet been localized cant difference detectable in cells incubated at 10ЊC
Proceedings of The National Academy of Sciences, 1986
The effect of temperature on secretory protein transport was studied by cell fractionation ofrat pancreatic lobules, pulse-labeled in vitro with [35S]methionine and chased for 60 min at 16, 20, or 370C. Chase at 370C allowed secretory proteins to reach a zymogen granule fraction, whereas chase at 16 or 20°C led to their extensive retention in a total microsomal fraction. To pinpoint the sites of transport inhibition, total microsomes were subfractionated by flotation in a sucrose density gradient. Five bands were resolved, of which the heaviest or Bi (density = 1.20 g/ml) consisted primarily of rough microsomes. The lighter fractions, B2 (1.17 g/ml), B3 (1.15 g/ml), and B4 (1.14-1.13 g/ml), consisted primarily of smooth vesicles derived from Golgi elements. B4 had the highest specific activity for galactosyltransferase, a trans Golgi cisternal marker; B2, B3, and B4 are assumed to represent cis, middle, and trans Golgi subcompartments, respectively. At the end of a 2-min pulse, a single peak of labeled proteins colocalized with B1. During subsequent 60-min chases, labeled proteins advanced to B2 at 160C and to B3 at 20°C. At 37C the radioactivity remaining in the total microsomal fraction was distributed among four peaks (B1-B4). The results indicate that transport from the endoplasmic reticulum to the Golgi complex is strongly inhibited below 200C. At 16°C, the bulk of the cohort of labeled secretory proteins is still in the rough endoplasmic reticulum, but its advancing front reaches cis Golgi elements. At 200C, the front advances to a middle Golgi compartment, and at 37PC most of the cohort (,u70%) reaches condensing vacuoles and zymogen granules. It is concluded that transport steps along the endoplasmic reticulum-plasmalemma pathway have distinct temperature requirements.
Diffusional Mobility of Golgi Proteins in Membranes of Living Cells
Science, 1996
the baculovirus expression vector pVL1 393 (Invitrogen) with a His tag at the COOH-terminal end. Proteins were purified by Ni-affinity chromatography (Qiagen). STATs 1, 5, and 6 were expressed and purified by identical methods. For DNA binding studies, insect cells were infected with recombinant viruses encoding JAK1 and one of the STATs. 10. T. K. Blackwell and H. Weintraub, Science 250, 1104 (1990). 11. The optimal STAT4 binding site was selected from a pool of oligonucleotides with random bases at 14 adjacent positions. The bound sequences were separated from the unbound by gel mobility-shift. After three rounds of selection the bound fragments were cloned, and 18 independent isolates were sequenced. All 18 fragments displayed the same 9-nucleotide consensus binding sequence, TTCCGGGAA.
Traffic, 2002
Golgi network that interacts with microtubule minus ends. GMAP-210 overexpression has previously been shown to perturb the microtubule network and to induce a dramatic enlargement and fragmentation of the Golgi apparatus (Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM. J Cell Biol 1999; 145: 83-98). We now report that overexpressing GMAP-210 blocks the anterograde transport of both a soluble form of alkaline phosphatase and the hemagglutinin protein of influenza virus, an integral membrane protein, between the endoplasmic reticulum and the cis/medial (mannosidase II-positive) Golgi compartment. Retrograde transport of the Shiga toxin B-subunit is also blocked between the Golgi apparatus and the endoplasmic reticulum. As a consequence, the B-subunit accumulates in compartments positive for GMAP-210. Ultrastructural analysis revealed that, under these conditions, the Golgi complex is totally disassembled and Golgi proteins as well as proteins of the intermediate compartment are found in vesicle clusters distributed throughout the cell. The role of GMAP-210 on membrane processes at the interface between the endoplasmic reticulum and the Golgi apparatus is discussed in the light of the property of this protein to bind CGN membranes and microtubules.