Structural integrity of the Golgi is temperature sensitive in conditional-lethal mutants with no detectable GM130.
At 39.5 degrees C in the temperature-sensitive, conditional-lethal mutant ldlG, glycoprotein processing is disrupted and secretion is blocked. The ultrastructure of the Golgi apparatus in ldlG cells was examined using immunofluorescence and immunoelectron microscopy. At 34 degrees C the structure of the Golgi apparatus was normal, whereas after incubation at 39.5 degrees C for 12 h it disassembled into dispersed vesicles. These reassembled into stacks when cells were returned to 34 degrees C for 6 h. At both 34 and 39.5 degrees C, all Golgi markers examined were present at wild-type levels except GM130, which was undetectable (<5% of control). Transfection with GM130 corrected the mutant phenotypes. Although the endogenous gene encoding NSF is apparently normal in ldlG cells, all mutant phenotypes were corrected by transfection with NSF, suggesting that NSF functioned as an extragenic suppressor. These findings provide additional support for a role of GM130 in determining the properties of the Golgi apparatus and for NSF in influencing GM130 stability and function. They also suggest that, at 34 degrees C, detectable levels of GM130 are not required for normal Golgi morphology and function, but that GM130 - or a GM130-dependent protein(s) - does play a role in protecting the Golgi, and thus the cells, from stress at higher temperatures.
Vasile
Eliza
E
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Perez
Tomas
T
Nakamura
Nobuhiro
N
Krieger
Monty
M
D000818:Animals; D001324:Autoantigens; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D005804:Genes, Lethal; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure*; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D013696:Temperature
D001324:Autoantigens; D018076:DNA, Complementary; C108705:GORASP1 protein, human; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins; D012260:Ribonucleases; C090042:gigantin