Endocytic trafficking of Gpm6a is altered by mutation of the key residue E258 critical for its function in filopodia formation

MONTIEL, M.B.; FUCHSOVA, B.; ROSAS, N.M.

San Luis

Congreso; XXXVIII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias (SAN); 2023

Gpm6a is a neuronal membrane glycoprotein that functions in the processes of neuronaldevelopment and its overexpression leads to the extensive formation of filopodia. Previously,we showed that Gpm6a lacking C- but not N-terminal cytoplasmic tail fails to inducefilopodium formation and we identified K250, K255, and E258 within the C-terminus as the keyfunctional residues in this process. We observed that Gpm6a lacking C-terminus and the pointmutation E258A, display high accumulation in the cytosol suggesting that cell surfacetrafficking is affected. Since different types of membrane outgrowth, filopodia formationincluding, require polarized membrane transport we hypothesized that the incapacity of themutant Gpm6a to induce filopodium formation could be linked to the disrupted trafficking ofmutant Gpm6a.Here, we show using confocal microscopy that the mutant forms of Gpm6a that fail toinduce filopodia formation display preferential localization to the Lamp1-positive structures.Our colocalization assays show that the wt Gpm6a and all studied mutant proteins clearlyprogress from the plasma membrane to Lamp1-containing late endosomal/ lysosomal structuresbut the Gpm6a ΔC-EGFP and E258-EGFP mutations result in increased accumulation ofGpm6a in these compartments. At the same time, the overexpression of E258-EGFP leads todecreased neuronal arborization as assessed by Sholl analysis and differences in thecolocalization of Gpm6a with Rab11.