Constitutive secretion in Tetrahymena thermophila

Abstract

The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates.

Fig. 1.

SDS-PAGE analysis of SCM from different strains (CU428, SB281, and SB210) under different culture conditions (growth to stationary phase in rich medium and starvation medium). Protein markers were run in lane 5, with standards at 27 and 66 kDa staining most strongly; molecular masses of standards are given at left.

Fig. 2.

Venn diagram indicating the intersection of proteins identified by ESI-MS from CU428 whole cells, proteins secreted by CU428 during growth to stationary phase in rich medium, and proteins secreted by CU428 during overnight starvation. The total number of proteins represented by each circle is shown by the value in the adjoining oblong.

Fig. 3.

Venn diagram indicating the intersection of proteins identified by ESI-MS in SCM of each strain cultured either in rich medium or the starvation condition, showing results for CU428 (A), SB210 (B), and SB281 (C). The total number of proteins represented by each circle is shown by the value in the adjoining oblong.

Fig. 4.

Venn diagram indicating the intersection of proteins identified by ESI-MS in SCM of each strain (CU428, SB210, and SB281), showing results from culture conditions of rich medium (A) and starvation medium (B). The total number of proteins represented by each circle is shown by the value in the adjoining oblong.

Fig. 5.

Functional groupings of secretome proteins detected by ESI-MS in SCM of each strain cultured under each condition. Populations are based on nPTPSI values determined by the program Spectrum Mill as described in Materials and Methods; numbers of individual proteins are listed in Table S2 in the supplemental material. Functional group assignments of individual proteins are listed in Table S4 in the supplemental material.