Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon-COP.
The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.
Guo
Q
Q
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Vasile
E
E
Krieger
M
M
D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D016466:CHO Cells; D003001:Cloning, Molecular; D020756:Coatomer Protein; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000502:physiology; D005459:Fluorides / Q000494:pharmacology; D005804:Genes, Lethal / Q000235:genetics*; D006056:Golgi Apparatus / Q000187:drug effects / Q000648:ultrastructure*; D006358:Hot Temperature; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017607:Aluminum Compounds; D020756:Coatomer Protein; D018076:DNA, Complementary; D008565:Membrane Proteins; D011973:Receptors, LDL; C050992:tetrafluoroaluminate; D005459:Fluorides