A portion of UCH-L1 is membrane-associated and farnesylated. (A) Rat brain synaptic vesicle fractions were treated with increasing salt concentrations, detergent (TX), or buffer only (Mock), as indicated. Proteins that dissociated from the vesicles were collected in supernatant fractions (S), and those that remained with the vesicular membranes were collected in the pellet fractions (P). The presence of synapsin I, synaptophysin, and UCH-L1 in each fraction was determined by Western blotting. Similar results were obtained with NaCl and KCl salts. (B) 2D electrophoresis followed by Western blot analysis using UCH-L1 antibody was performed on recombinant UCH-L1 protein (i); on the membrane fraction (ii) and whole lysate (iii) of rat brain; on lysates of SH-SY5Y cells treated with DMSO (iv) or 100 nM FTI-277 (v); and on H1299 cell lysate (vi). (C) Two-dimensional analysis was performed as in B on lysates of COS-7 cells transfected with WT (i), CVIM (ii), or C220S UCH-L1 (iii); and on lysates of WT UCH-L1-transfected cells treated with DMSO (iv) or 100 nM FTI-277 (v). Closed arrowheads indicate forms of UCH-L1 that have the same pI value as the recombinant protein; open arrowheads indicate species corresponding to the form of UCH-L1 found in the membrane fraction. (D) COS-7 cells expressing either WT or C220S FLAG-tagged UCH-L1 were radiolabeled with [³H]farnesol and immunoprecipitated by using anti-FLAG antibody. Eluted proteins were analyzed by radiography and by Western blotting for UCH-L1.

α-Synuclein accumulation and toxicity are increased by farnesylated UCH-L1. (A and B) COS-7 cells were transfected with α-synuclein plus either vector or UCH-L1 variants, as indicated. At 72 h later, protein levels were determined by Western blotting of cell lysates and densitometric analysis. α-Synuclein levels were normalized to actin and plotted relative to vector control. (C) SH-SY5Y cells were cotransfected with α-synuclein (Syn) and N-terminally Flag-tagged UCH-L1 (WT or C220S), as indicated. Cell lysates were analyzed as above. In parallel, cell viability was assessed by flow cytometry based on cell size, complexity, and membrane integrity. Cell numbers were normalized against control cells transfected with α-synuclein and vector; n = 3. Data represent mean ± SE throughout (#, P < 0.05; *, P < 0.005; **, P < 0.001).

UCH-L1 associates with the endoplasmic reticular membrane. (A) COS-7 cells were transiently transfected with either GFP or GFP-UCH-L1, as indicated. Cells were fixed in paraformaldehyde (PF) or alcohol, and GFP fluorescence was imaged by using identical exposure times and image settings. Alcohol fixation releases cytosolic proteins, including free GFP, from cells. (B) Fluorescence microscopy was used to image COS-7 cells transfected with GFP-UCH-L1 and immunostained with calreticulin antibody (i), as well as SH-SY5Y (ii) and H1299 (iii) cells immunostained with UCH-L1 and calreticulin antibodies. COS-7 and SH-SY5Y cells were fixed in alcohol; H1299 cells were fixed in paraformaldehyde. Green indicates UCH-L1; red, calreticulin; yellow, colocalization; and blue, DAPI nuclear stain. (Scale bar: 10 μm.)

An FTase inhibitor reduces α-synuclein accumulation and toxicity by a mechanism requiring the expression and activity of UCH-L1. (A) Cell viability was measured by MTT assay in SH-SY5Y cells infected with control virus (Con), virus expressing LacZ, or virus expressing α-synuclein (Syn) and treated with either DMSO or 100 nM FTI-277 (n = 3; *, P < 0.05). Values represent mean ± SE throughout. (B) α-Synuclein levels were quantified by Western blotting and normalization to actin in SH-SY5Y cells infected with α-synuclein-expressing virus and treated with either DMSO or 100 nM FTI-277 (n = 3; *, P < 0.05). (C) SH-SY5Y cells were transfected with either nonspecific (NS) or UCH-L1-specific siRNA oligos, followed by infection with α-synuclein-expressing virus and treatment with either DMSO or 100 nM FTI-277. Cell viability was assessed by flow cytometry and normalization against control cells that had undergone the same treatment, except that they were infected with vector instead of α-synuclein. UCH-L1 levels were analyzed by Western blot in a portion of cells before infection (n = 4; *, P < 0.01). (D) SH-SY5Y cells infected with α-synuclein-expressing virus were treated with 100 nM FTI-277, LDN-57414 (LDN), or their combination. Viable cells were counted by trypan blue staining (n = 3; *, P < 0.05) (E) Primary midbrain cultures were infected with either vector (Vec) or α-synuclein-expressing virus (Syn) and treated with DMSO or 100 nM FTI-277. Following treatment, tyrosine hydroxylase-positive (TH+) neurons were counted by using immunofluorescence microscopy. Four fields were counted and averaged in each of 3 independent experiments (n = 3; *, P < 0.01). (F) Endogenous α-synuclein levels were quantified as in B in cultured primary cortical neurons treated with either DMSO or 100 nM FTI-277 (n = 4; *, P < 0.05). Tubulin was used for normalization.