Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation.
The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.
Chilcoat
N D
ND
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637, USA.
Melia
S M
SM
Haddad
A
A
Turkewitz
A P
AP
D000143:Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D015800:Protozoan Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000648:ultrastructure; D014158:Transcription, Genetic
D000143:Acids; D002135:Calcium-Binding Proteins; C103333:GRL1 protein, Tetrahymena thermophila; D015800:Protozoan Proteins