The small number of docked mucocysts that accumulate in cells may be accounted for by the fact that we could not disrupt all macronuclear copies of the apparently essential gene. Significantly, cells are deficient in the processing of all Grl proteins tested. threonine proteases (Eisen Gene Expression Database (TGED; http://tged.ihb.ac.cn/) subsequently reorganized at the Functional Genomics Database (TetraFGD; http://tfgd.ihb.ac.cn/), to ask whether any putative proteases are also coregulated with genes. We identified four cathepsins (genes (Figure 1A) but distinct from those of other, closely related proteases (Figure 1B). Open in a separate window FIGURE 1: Expression profiling identifies a set of enzymes that are coregulated with secretory Mephenesin granule cargo genes. (A) The expression profiles of four predicted cathepsin genes (and Functional Genomics Database. In the plots here, each trace was normalized to that gene’s maximum expression level. The culture conditions sampled at successive time points represent growing (L-l, L-m, and L-h), starved (S-0, S-3, S-6, S-9, S-12, S-15, and S-24), and conjugating (C-0, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16, and C-18) cultures. Details on the sampling times are found in Miao belongs to the cysteine-protease subgroup (cathepsin C family). Another cysteine protease, cathepsin B, was previously studied in and shown to localize to food vacuoles (Jacobs aspartyl proteases and aspartic proteases (Supplemental Figure S1). The cysteine proteases possess conserved triad catalytic residues (C, H, N; Figure 1C). The Car1p sequence contains a putative catalytic glutamate (E) at an appropriate position, but this is weakly determined, given the minimal size of this motif and the limited overall sequence identity with characterized carboxypeptidases in other nonciliate species. The phylogenetic relationships between the aspartyl proteases and a set of related enzymes from other eukaryotes are shown in Figure 2. The aspartyl cathepsins fall within a cluster of genes from ciliates and the related apicomplexan parasites (Figure 2). The carboxypeptidase has close homologues only in other ciliates (and species, and aspartyl cathepsins are in boldface. Blocks of cathepsins that fall within single lineages or a group of closely related lineages are shown in color SOCS-2 blocks (see color key at bottom of figure). (Tg), spp. (P), spp. (Hs), (Tt), (Pt), (Im). See Supplemental Table S3 for a list of accession numbers for all sequences. Gene disruption implicates each of the aspartyl cathepsins in mucocyst biogenesis, with a special role for macronuclei (Karrer, 2000 ). If a gene is essential for cell viability, one cannot recover daughters in which all intact macronuclear copies have been replaced. Open in a separate window FIGURE 3: Disruption of and and genes by the Neo4 drug resistance cassette were targeted by homologous recombination. A detailed description of the construction and use of the and knockout constructs is in were run in parallel. To assess the extent of gene expression, we used reverse transcription PCR (RT-PCR) to monitor the knockout strains. For knockout showed an increase in doubling time, confirmed for multiple clones (Supplemental Table S1), indicating that may be important for growth under these culture conditions. To ask whether any of these genes is involved in mucocyst secretion, we first tested the exocytic response using a semiquantitative assay based on stimulation by dibucaine, which triggers mucocyst exocytosis (Satir, 1977 ; Cowan mutant was identical to wild type (Figure 4B, iv, right ). The and mutant strain (Figure 4B, iii, right). For that reason we focused further studies on the gene, using the and cathepsin B (mutants show reduced release of mucocyst Mephenesin Mephenesin contents. (A) Cartoon representing a semiquantitative assay for mucocyst discharge. Cells are stimulated with dibucaine for 20 s, which triggers regulated exocytosis of mucocyst contents. Subsequent centrifugation results in a dense pellet of cells, with an overlying flocculent composed Mephenesin of expanded dense cores of exocytosed mucocysts. (B) Four independent stimulated wild-type (WT) cell cultures generate the expected two-layered pellet (iCiv, left). For clarity, the flocculent layer in this and all samples are delineated with a dashed line at the lower border and an unbroken line at the upper border. Stimulated cells produce a flocculent.