Abstract
Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.
Highlights
Cells can respond to and shape their environments by taking up and releasing macromolecules
One family of key determinants are monomeric GTPases called Rabs, which function as molecular switches by interacting with membrane bilayers and diverse protein effectors in cycles controlled by GTP binding and hydrolysis [5]
A fundamental aspect of Rab function is that multiple Rabs, encoded as a gene family, are co-expressed within a single cell, and the individual family members are each targeted to a small subset of membrane compartments, where they interact with unique effectors [6]
Summary
Cells can respond to and shape their environments by taking up and releasing macromolecules. Traffic through the network is highly regulated, and a convergence of data from structural, functional, and evolutionary studies demonstrate that gene families encode proteins functioning as conserved specificity determinants for endocytic and exocytic compartments [2,3] They do so primarily by controlling the formation, targeting and fusion of vesicles that transport cargo between compartments [4]. The Rab clade has remained highly conserved at the sequence level among all eukaryotic kingdoms; the corresponding proteins, where they have been characterized, are all associated with traffic between the endoplasmic reticulum and cis-Golgi [10] These experimental findings coupled with phylogenetic parsimony reveal that Rab was already a determinant for this step in an early eukaryotic ancestor. It follows that the analysis of lineage-specific expansions or losses in conserved Rab clades can provide insights into the range of evolutionary paths that have led to modern cells
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