Molecular mechanisms of inner nuclear membrane protein turnover

Molecular mechanisms of inner nuclear membrane protein turnover The nuclear envelope of eukaryotic cells consists of two concentric membrane layers, the inner (INM) and the outer (ONM) nuclear membrane, and nuclear pore complexes (NPCs) forming transport channels through the double membrane system. The ONM is continuous with the endoplasmic reticulum (ER) membrane. Although ONM and INM are connected at the sites of NPCs, they contain different sets of integral membrane proteins. Proteomic studies have identified over 60 putative INM proteins, out of which a dozen have been characterized and found to function in genome organization and gene expression. To reach the INM, integral membrane proteins synthesized at the ER diffuse laterally within the ER- ONM to the cytoplasmic side of NPCs, where they have to pass the physical barrier of the NPC at the pore membrane. Several models have been proposed for nuclear targeting of INM proteins, including i) diffusion in the pore membrane through the peripheral NPC channels followed by retention at the INM, and ii) a nuclear localization signal (NLS)-mediated active transport through the peripheral channels. While nuclear targeting of INM proteins has been studied extensively, their destiny after correct targeting remains elusive. Due to nuclear compartmentalization and seclusion of the INM from the rest of cellular membrane networks by the NPC-barrier, INM protein turnover is an intriguing, though poorly addressed biological question. Are INM proteins transported back to the cytoplasm to be degraded by cytoplasmic and ER-associated machineries, or are they degraded by pathways located in the nucleus? To address these questions we will study several integral INM proteins in yeast Saccharomyces cerevisiae, Asi1, Asi2, Asi3 and Src1, and the mammalian Src1 ortholog, LEM2. In pulse chase experiments we will determine INM protein half-lives and examine involved degradation pathways, taking advantage of yeast mutant strains defective in either proteasomal and ER-associated degradation, or in lysosomal protein degradation. In addition, we will determine the spatial organization of the INM protein degradation by using yeast mutant strains containing cytoplasmically-anchored or exclusively nuclear components of the degradation pathway. We will also test the regulation of INM protein degradation by ubiquitination and will identify degradation targeting domains in INM proteins by testing truncation mutants. Finally, turnover of mammalian LEM2 will be studied in fibroblasts and in in vitro myoblast differentiation models. This will allow testing i) whether degradation of LEM2 and yeast Src1 involves similar pathways, and ii) whether LEM2 turnover is regulated during the cell cycle and muscle differentiation. Overall this study is expected to provide the basis for understanding the biological process of INM protein turnover. Furthermore, since protein degradation is important in protecting cells from the accumulation of aberrant proteins, this study will also yield new insights into human diseases linked to formation of aberrant protein aggregates in the nucleus.

Proteins are major biomolecules in our cells involved in the generation of cellular structures and organelles and in nearly all biochemical cellular processes. Cells in our body constantly produce new proteins, which have to be correctly folded and transported to specific cellular compartments to function correctly. Due to errors in the folding process and due to damage of proteins through internal and external chemical processes and toxic compounds, proteins have a limited 'life time' and have to be replaced (turned over) by new fully functioning proteins. It is essential for a cell to dispose misfolded and damaged proteins in a regulated manner, as accumulation and aggregation of these non-functional proteins is linked to several human diseases. Therefore, an intricate cellular machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been identified and characterized in the cytoplasm of the cells, where they target and degrade misfolded proteins in the cytosol and in cytoplasmic membrane structures, such as the endoplasmic reticulum. The latter pathway involves many tightly regulated components and is called endoplasmic reticulum associated degradation (ERAD). However, similar pathways that can remove damaged proteins in the membrane of the cell nucleus have not been described yet and it remained unclear, if and how nuclear membrane proteins are turned over, despite the fact that the accumulation of damaged proteins in the nucleus is linked to several neurodegenerative diseases and to the premature ageing disease Hutchinson Gilford Progeria Syndrome. In this project, using mainly yeast as an experimental system because of the lower complexity of yeast protein degradation pathways, we identified the first protein degradation machinery localized in the nucler membrane that targets nuclear membrane proteins. Interestingly, these components were previously characterized and described to function in the cytoplasmic ERAD machinery. Thus our study showed for the first time that components of the cytoplasmic ERAD pathway function also in the nucleus to degrade nuclear proteins. In addition, we identified a second degradation pathway in the nucleus that seems to involve nuclear-specific proteins, leading to the new emerging concept that the nucleus and in particular the nuclear membrane is a major site in the cell for protein quality control, and to the definition of inner nuclear membrane associated degradation (INMD) pathways, which share several components with cytoplasmic ERAD pathways, but are cleary different from ERAD based on the different types of proteins they degrade. Overall our study identified the first degradation-linked protein quality control pathway located in the nucleus and therfore has important implications for the development of therapeutic drugs for all human diseases linked to nuclear protein aggregation.