The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins.