Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.
The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.
Bowman
G R
GR
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Turkewitz
A P
AP
D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002499:Centrifugation, Density Gradient; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004843:Epistasis, Genetic; D019084:Fluorescent Antibody Technique, Indirect; D005821:Genetic Techniques; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016808:Tetrahymena thermophila / Q000235:genetics*; D013997:Time Factors
D003513:Cycloheximide