Abstract
Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1). While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.
Highlights
Endocytosis is conserved in eukaryotes, but the molecular machinery deployed by cells to internalize plasma membrane varies according to task, which can range from nutrient absorption to cell signaling [1]
Clathrin Is Essential for FM1–43 Uptake To determine whether FM1–43-labeled vesicles reflected clathrin-mediated endocytosis (CME) in this system, we identified a single clathrin heavy chain (CHC) ortholog (CHC1) in the Tetrahymena macronuclear genome
We initially focused our analysis on unambiguously aligned regions in the GTPase domain, middle domain, and GTPase effector domain (GED) common to all dynamins and dynamin-related protein (DRP). (For domain boundary definitions, see Materials and Methods and Figure S1.) The phylogeny generally confirmed a clustering of proteins according to function, as expected (Figure 8)
Summary
Endocytosis is conserved in eukaryotes, but the molecular machinery deployed by cells to internalize plasma membrane varies according to task, which can range from nutrient absorption to cell signaling [1]. The best understood mechanism of endocytosis involves clathrin-induced membrane deformation to form nascent vesicles (Figure 1A) [2]. Clathrin can recruit membrane proteins for internalization, often via a multimeric adaptor protein (AP) complex, AP-2 [3]. Clathrin-mediated endocytosis (CME) requires classical dynamin, a member of a family of selfassembling GTPases. The GTP-dependent constriction of an oligomeric dynamin ‘‘collar’’ may induce fission of vesicles from plasma membrane [4,5]. Dynamin recruits effectors, including actin-binding proteins, which could mediate aspects of vesiculation [6,7]
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