Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila.
Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation-a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation.
Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637.
Cassandra
C
Kontur
Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637.
Santosh
S
Kumar
Department of Genetics, Stanford University, California 94305 Howard Hughes Medical Institute, Stanford University, California 94305.
Xun
X
Lan
Department of Genetics, Stanford University, California 94305 Howard Hughes Medical Institute, Stanford University, California 94305 Department of Biology, Stanford University, California 94305.
Jonathan K
JK
Pritchard
Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637 apturkew@uchicago.edu.
Aaron P
AP
Turkewitz
D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis / Q000235:genetics; D005786:Gene Expression Regulation; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D016296:Mutagenesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000235:genetics* / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000502:physiology
D011506:Proteins
dense core; endomembranes; evolutionary cell biology; regulated exocytosis; secretion