Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. Secretion of glycosylated tripeptide and truncated sphingomyelin.
The constitutive secretory pathway has been reconstituted in mechanically permeabilized Chinese hamster ovary cells using two secretory markers, an acyltripeptide (N-octanoyl-Asn-Tyr-Thr-NH2) that is glycosylated at Asn in the endoplasmic reticulum and a truncated ceramide that is converted to sphingomyelin. Secretion of these bulk phase markers is dependent on cytosolic proteins and ATP. Secretion of both the glycosylated tripeptide and truncated sphingomyelin was inhibited at 15 degrees C. These results are taken as evidence that the vesicle flow to the plasma membrane (rather than artificial lysis of endoplasmic reticulum or Golgi cisternae) is required for the release of markers to the medium. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a nonhydrolyzable analogue of GTP, inhibited secretion, resulting in an accumulation of both the glycosylated tripeptide and truncated sphingomyelin in the semi-intact cell. Inhibition of secretion by GTP gamma S was not observed in the presence of the aminoglycoside antibiotic neomycin.
Helms
J B
JB
Center for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands.
Karrenbauer
A
A
Wirtz
K W
KW
Rothman
J E
JE
Wieland
F T
FT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D001921:Brain / Q000648:ultrastructure; D002417:Cattle; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003600:Cytosol / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008099:Liver / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism*
D009842:Oligopeptides; D013109:Sphingomyelins; C066475:N-octanoyl-asparaginyl-tyrosyl-threoninamide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D009355:Neomycin