New roles for ancient interactions: functional analysis of the conserved interactions between the clathrin heavy and light chains and calmodulin

Abstract

Clathrin is an evolutionary conserved molecule that coats membranes endocytosed from the plasma membrane and those that move between the trans-Golgi network (TGN) and endosomes of eukaryotic cells. It also plays roles in early stages of mitosis, cytokinesis, chromosome alignment and in certain phagocytic events. At the cellular level, clathrin is required for major regulated secretory and endocytic pathways, which transport proteins, lipids, soluble factors, and extracellular ligands between the plasma membrane and the intracellular compartments. In multicellular organisms clathrin is involved in numerous specialized pathways of physiological relevance, such as the production of morphogen gradients, the formation of secretory granule, antigen presentation by the immune system, virus maturation, the control of glucose homeostasis, or synaptic vesicle generation. Many proteins that interact with clathrin have been identified. In some cases the functional relevance of these molecular interactions remains unknown. Among those, the clathrin-calmodulin interaction is probably one of the most striking examples since it involves two evolutionary conserved essential proteins and it was detected in vitro decades ago. However, it was not clarified whether clathrin and calmodulin interacted in vivo and what was the functional significance of this interaction. The calcium sensor calmodulin plays an important role regulating a wide variety of proteins and important signaling pathways in all eukaryotes. It modulates the activity of key regulatory enzymes, ion pumps and proteins implicated in motility and it has essential roles in mitosis. Importantly, it has also been found to be implicated in endocytosis, membrane traffic and endosome fusion. Nevertheless, most targets of calmodulin and the mechanisms by which calmodulin regulates specific membrane transport events are mainly unknown.

The objective of this Thesis is to address the study of the clathrin-calmodulin interaction, whose functional significance has not been studied thus far, using the yeast Saccharomyces cerevisiae as a model system. S. cerevisiae has proven to be an extremely versatile organism for studying many cellular processes, because of the ease of combining biochemical analyses with genetic studies and live imaging in this organism and the evolutionary conservation of most essential eukaryotic cellular processes and proteins involved.

In this study, we reveal that the clathhrin heavy and light chains bind calmodulin not only in vitro but also in yeast, most likely in a calcium-dependent manner. Further, we describe a not-previously­characterized calmodulin binding site at the C-terminus of the clathrin heavy chain, and identify calmodulin point mutations that specifically disrupt their interaction with clathrin. Finally, we provide evidence supporting a specific role of the clathrin-calmodulin interaction in the retrograde transport from endosomes to the TGN.

La clatrina es una molécula evolutivamente conservada que envuelve las membranas que se endocitan desde la membrana plasmática y aquellas que se mueven entre la red trans-Golgi (TGN) y los endosomas de las células eucariotas, en las principales vías celulares reguladas secretoras y endocíticas. Muchas proteínas que interactúan con clatrina han sido identificadas, pero en algunos casos se desconoce la relevancia funcional de estas interacciones. Entre ellas, la interacción de la clatrina con la calmodulina, dos proteínas esenciales evolutivamente conservadas, fue detectada in vitro hace décadas pero no se sabe si interactúan in vivo y cuál es el significado funcional de esta interacción. La calmodulina es un sensor de calcio que juega un papel importante la regulación de una amplia variedad de proteínas y rutas de señalización importantes en todos los organismos eucariotas. Es importante destacar que también se ha visto implicada en la endocitosis, el tráfico de membranas y la fusión de endosomas. Sin embargo, muchas de las dianas de calmodulina y los mecanismos por los que la calmodulina regula eventos específicos de transporte de membranas son desconocidos.

En esta Tesis se aborda el estudio de la interacción clatrina-calmodulina, cuyo significado funcional no se ha estudiado hasta el momento, utilizando la levadura Saccharomyces cerevisiae como sistema modelo. En el estudio, revelamos que las cadenas pesada y ligera de clatrina se unen a calmodulina no sólo in vitro sino también en levadura, probablemente de una forma dependiente de calcio. Además, describimos un sitio de unión a calmodulina que no estaba previamente caracterizado, en el extremo C-terminal de la cadena pesada de clatrina, e identificamos mutaciones puntuales en calmodulina que alteran específicamente su interacción con la clatrina. Por último, aportamos pruebas que respaldan una función específica de la interacción clatrina-calmodulina en el transporte retrógrado desde los endosomas al TGN.