FMRP shows a diverse subcellular distribution pattern in HeLa cells as revealed by subcellular fractionation analysis.

<p>(A) Experimental cell fractionation procedure employing several differential centrifugation steps. Cells were fractionated into six distinct fractions, including heavy membrane (plasma membrane and rough endoplasmic reticulum), light membrane (polysomes, golgi apparatus, smooth endoplasmic reticulum), cytoplasm (cytoplasm and lysosomes), enriched nuclear membrane (containing rough endoplasmic reticulum), nucleoplasm, and nucleoli. S, supernatant; P, pellet. (B) FMRP is largely absent in the cytoplasm and nucleoplasm and predominantly localizes to solid compartments. The protein concentrations were normalized in all fractions with exception of the nucleoplasm due its low protein content as compared to the other fractions. In each lane, 5 μg proteins were loaded except for the nucleoplasm, where one μg was used. In addition to FMRP and its binding partner CYFIP, the fractions were analyzed by using different subcellular marker, including Gα_q/11, Na⁺/K⁺-ATPase and Rac1 (plasma membrane), EEA2 (endosomes), GAPDH (cytoplasm), eIF5 and RPLP0 (ribosomes and rough ER). Nuclear markers included histone H3 and lamin B1. Nucleolin was used as nucleolar marker. (C) Detection of FMRP in mitochondria. The presence of FMRP in isolated mitochondrial fraction was analyzed by SDS-PAGE and immunoblotting, using antibodies against FMRP, two mitochondrial proteins MTCO2 and ACAT1, the cytosolic GAPDH as well as the nuclear proteins lamin B1, histone H3 and nucleolin. Equal protein amounts of the mitochondrial fraction and the total cell lysate were used.