Jerry E. Honts

A Proteomics Approach to Cloning Fenestrin from the Nuclear Exchange Junction of Tetrahymena

ABSTRACT. We set out to find the “fenestrin” gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS‐PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP‐localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.

Nested Genes CDA12 and CDA13 Encode Proteins Associated with Membrane Trafficking in the Ciliate Tetrahymena thermophila

ABSTRACT We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense CDA12 RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense CDA13 RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate T. thermophila. We also highlight an unusual aspect of genome organization in Tetrahymena, namely, the existence of nested, antisense genes.

Novel Cytoskeletal Proteins in the Cortex of Tetrahymena1

Abstract An important unsolved problem lies in the mechanisms that determine overall size, shape, and the localization of subcellular structures in eukaryotic cells. The membrane skeleton must play a central role in these processes in many cell types, and the ciliate membrane skeleton, or epiplasm, offers favorable opportunities for exploring the molecular determinants of cortical organization. Among the ciliates, Tetrahymena is well suited for the application of a wide range of molecular and cellular approaches. Progress has been made in the identification and sequencing of genes and proteins that encode epiplasmic and cortical proteins. The amino acid sequences of these proteins suggest that they define new classes of cytoskeletal proteins, distinct from the articulin and epiplasmin proteins. We will also discuss recent in vivo and in vitro studies of the regulation of assembly of these cortical proteins. This will include information regarding the down-regulation of epiplasmic proteins during cleavage, their topographic regulation in the cell cycle, and the results of in vitro assembly and binding studies of the epiplasmic C protein.

Purification of Tetrahymena Cytoskeletal Proteins

Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

Solution NMR structures of the C-domain ofTetrahymenacytoskeletal protein Tcb2 reveal distinct calcium-induced structural rearrangements: NMR Structures of Apo- and Ca2+-Tcb2-C

Tcb2 is a calcium‐binding protein that localizes to the membrane‐associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium‐triggered, ATP‐independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2s structure‐function relationship and contractile properties, we determined solution NMR structures of its C‐terminal domain in the calcium‐free and calcium‐bound states. The overall architecture is similar to other calcium‐binding proteins, with paired EF‐hand calcium‐binding motifs. Comparison of the two structures reveals that Tcb2‐Cs calcium‐induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full‐length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748–1756.

Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from Tetrahymena thermophila

Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. 1H–15N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.