High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein-protein or protein-membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells.

Not Just Another Scaffolding Protein Family: The Multifaceted MPPs.

Membrane palmitoylated proteins (MPPs) are a subfamily of a larger group of multidomain proteins, namely, membrane-associated guanylate kinases (MAGUKs). The ubiquitous expression and multidomain structure of MPPs provide the ability to form diverse protein complexes at the cell membranes, which are involved in a wide range of cellular processes, including establishing the proper cell structure, polarity and cell adhesion. The formation of MPP-dependent complexes in various cell types seems to be based on similar principles, but involves members of different protein groups, such as 4.

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane.