Expression profiling suggests a role for sortilin-family receptors in mucocyst biogenesis in T. thermophila. (A) Sortilins are coregulated with genes encoding mucocyst contents in T. thermophila. The expression profiles of the four T. thermophila sortilins (SOR1–4; right), are similar to those of genes (GRL1, GRL3, GRT1, and IGR1; left) encoding mucocyst cargo proteins. Expression profiles are derived from the Tetrahymena Functional Genomics Database, with each profile normalized to that gene’s maximum expression level. Points on the x axis correspond to successive time points and represent growing, starved, and mating cultures, including three different culture densities (low [Ll], medium [Lm], and high [Lh]), 7 samples taken during 24 h of starvation, and 10 samples subsequently taken during 18 h after conjugation. (B) Expansion of the sortilin family in ciliates. The maximum likelihood tree illustrates a phylogeny of VPS10 domain–containing receptors (sortilins) in alveolates, the taxonomic group consisting of ciliates, apicomplexans, and dinoflagellates. Two of the T. thermophila sortilins, marked by black circles, cluster with the sortilins from other alveolates. In contrast, T. thermophila SOR2 and SOR4, marked by maroon diamonds, belong to an expansion of sortilins restricted to ciliates. Species are abbreviated as follows: Babesia microti (Bm), Cryptosporidium hominis (Ch), Cryptosporidium muris (Cm), I. multifiliis (Im), Neospora caninum (Nc), P. tetraurelia (Pt), Perkinsus marinus (Pm), Plasmodium berghei (Pb), Plasmodium cynomolgi (Pc), Plasmodium falciparum (Pf), Plasmodium knowlesi (Pk), Plasmodium vivax (Pv), Plasmodium yoelii yoelii (Py), T. thermophila (Tt), Theileria annulata (Ta), Theileria orientalis (To), Theileria parva (Tp), T. gondii (Tg). See Tables S1 and S2 for a list of accession numbers for all of the sequences. (C) Verification of the nonessential sortilin knockouts. cDNA was prepared from wild-type, Δsor1, Δsor2, and Δsor4 cells, and the SOR1, SOR2, and SOR4 sequences were PCR amplified using gene-specific primers. As shown in this 1% ethidium bromide–stained agarose gel, each of the gene knockout lines lacks the amplified product corresponding to the targeted gene, but shows wild-type levels of the other transcripts that serve as loading controls (see also Fig. S2 E). The lanes shown are all part of a single gel, but their order has been rearranged for this figure.