Caracterización funcional de la proteína HERC1

Abstract

INTRODUCTION. The human HERC family consists of six proteins whose sequences contain both HECT (Homologous to E6AP C-terminus) and RCC1-like domains (RLDs). While HECT domains are known to act as E3 ubiquitin ligases, RLDs behave, in at least some cases, as guanine nucleotide exchange factors for small GTPases. HERC1 is a giant (532 kDa) protein which has been implicated in membrane trafficking pathways through its ability to interact with clathrin heavy chain (CHC) as well as ARF- and Rab-family GTPases. OBJECTIVES. In this study, we sought to (i) identify new HERC1-binding molecules, (ii) pinpoint HERC1's subcellular localization, (iii) analyze the effects of siRNA-mediated HERC1 knockdown and (iv) characterize the behavior of truncated and mutant versions of HERC1. METHODS. HERC1-interacting molecules were found using yeast two-hybrid screens, pull-down, coimmunoprecipitation and lipid-protein overlay assays. The interactions were further characterized using ubiquitination and nucleotide exchange assays, gel filtration chromatography and enzyme activity measurements. The subcellular distribution of HERC1 was studied by confocal epifluorescence microscopy in several cell lines, which were also used to determine the effects upon intracellular trafficking pathways of a HERC1-specific siRNA and of a number of chimeric proteins between the green fluorescent protein (GFP) and various truncated and mutant forms of HERC1. RESULTS. M2-type pyruvate kinase (M2PK) was shown to interact with HERC1. However, HERC1 does not influence M2PK enzyme activity, oligomeric structure or ubiquitination. Clathrin light chain (CLC) and ARF6 also bind HERC1. Moreover, HERC1 stimulates GDP dissociation from ARF6, which, in turn, requires the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) bound to HERC1. Other phospholipids apart from PIP2 also display affinity for HERC1. Regarding its subcellular localization, HERC1 appeared partly in Golgi-associated vesicles and was seen to translocate to actin-rich membrane protrusions in an ARF6-dependent manner. On the other hand, HERC1 siRNA was shown not to affect endocytic or secretory pathways whereas, by contrast, some GFP-HERC1 chimeras cause alterations in endocytosis and the actin cytoskeleton. CONCLUSIONS. M2PK, CLC, ARF6 and several phosphoinositides have been identified as HERC1-binding molecules. HERC1 is Golgi-associated and probably has a regulatory function in membrane trafficking and/or in cytoskeletal rearrangements. "