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doi: 10.1242/10.1242/jcs.00617

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Lipid- and protein-mediated multimerization of PSD-95: implications for receptor clustering and assembly of synaptic protein networks

Karen S. Christopherson^1,2,*, Neal T. Sweeney^1,3,*, Sarah E. Craven^1,4, Rujun Kang⁵, Alaa El-Din El-Husseini^1,5 and David S. Bredt^1,

¹ Department of Physiology University of California at San Francisco, San Francisco, CA 94143-2140, USA
^* These authors contributed equally to this work

View larger version (20K): [in a new window]   Fig. 1. PSD-95 forms non-covalent multimers. COS cells were transfected with plasmids encoding GFP- and FLAG-tagged PSD-95 proteins as indicated. Cells were lysed with either 1% Triton X-100 or 0.1% SDS and proteins were immunoprecipitated with an antibody to GFP. Western blotting reveals that PSD-95/FLAG co-immunoprecipitated in cells lysed with Triton X-100 but not in SDS-treated lysates.

View larger version (26K): [in a new window]   Fig. 2. The N-terminal 13 amino acids of PSD-95 can mediate homomultimerization. COS cells were transfected with plasmids as indicated. Cell lysates were immunoprecipitated with antibodies to GFP or to the PDZ domains of PSD-95 as indicated and western blotting was used to monitor co-immunoprecipitation. (A) FLAG-tagged full-length PSD-95 and amino acids 1-13 of PSD-95 fused to GFP efficiently co-immunoprecipitated. (B) COS cells were co-transfected with constructs encoding PSD-95-GFP and amino acids 1-13 of PSD-95 fused to GFP. Co-immunoprecipitation of these constructs is disrupted when cells are lysed in buffer containing 0.1% SDS. (C) Chimeras of GFP and maltose binding protein (MBP), each containing the first 13 amino acids of PSD-95, efficiently co-immunoprecipitate.

View larger version (51K): [in a new window]   Fig. 3. Multimerization is a specific property of the PSD-95 palmitoylation motif. COS cells were transfected with plasmids encoding plasmids as indicated. Cell lysates were immunoprecipitated with an antibody to GFP, FLAG or the PDZ domains of PSD-95 as indicated and western blotting was used to monitor co-immunoprecipitation. (A) Mutating palmitoylated cysteine residues 3 and 5 of PSD-95 blocks co-immunoprecipitation. (B) Mutating leucine 4 of PSD-95 to serine (which blocks palmitoylation) disrupts co-immunoprecipitation. (C) Treating PSD-95-transfected COS cells for 4 hours with 20 µM 2-bromopalmitate – but not palmitate – disrupts multimerization mediated by the N-terminus of PSD-95. (D) Differently tagged chimeras of PSD-95 (containing just the palmitoylated N-terminal ten amino acids of GAP-43 fused to PSD-95 lacking a palmitoylation motif) do not oligomerize.

View larger version (83K): [in a new window]   Fig. 4. Ion channel clustering requires interaction of PSD-95 with a tetrameric ion channel. (A,B) PSD-95 forms clusters when co-transfected with K+ channel Kv1.4. (C,D) A tandem construct containing four covalently linked Kv1.4 subunits does not form clusters with PSD-95. (E,F) A monomeric transmembrane protein containing a C-terminal PDZ binding site (Tac-SDV) does not cluster when simply co-transfected with PSD-95. (G,H) However, this Tac-SDV construct is recruited to clusters when co-transfected with PSD-95 and Kv1.4. (I,J) This co-clustering requires the PDZ binding site on Tac, as the native Tac construct remains diffuse in cells containing PSD-95/Kv1.4 clusters. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 5. PSD-95/ion-channel clusters do not require PSD-95 multimerization but are reversibly dispersed by blocking palmitoylation of PSD-95. (A-D) PSD-95 clusters normally with a monomeric chimera containing the palmitoylated N-terminus of GAP-43 fused to PSD-95. (E,F) Incubating PSD-95/Kv1.4 co-transfected COS cells for 4 hours with 2-bromopalmitate disrupts PSD-95 ion channel clusters and causes both proteins to accumulate in perinuclear aggregates that resemble those formed (G,H) in cells co-transfected with Kv1.4 and the palmitoylation-deficient mutant of PSD-95 (C3,5S). (I,J) Treatment with palmitate as a control does not disrupt PSD-95/ion-channel clusters. (K,L) Somewhat smaller clusters are observed when Kv1.4 is co-transfected with a palmitoylation-deficient PSD-95 mutant that contains the paralemmin (PSD-95-prenyl) C-terminal consensus sequence for isoprenylation. (M,N) A 4-hour treatment with 20 µM 2-bromopalmitate does not disrupt ion channel clustering by PSD-95-prenyl. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 6. Prenylated PSD-95 does not multimerize with itself or with palmitoylated PSD-95. COS cells were transfected with plasmids encoding GFP- and FLAG-tagged PSD-95 constructs, some of which contain the paralemmin (PSD-95-prenyl) C-terminal consensus sequence for isoprenylation. Cells were lysed with 1% Triton X-100 and proteins were immunoprecipitated with an antibody to GFP. Western blotting reveals that PSD-95-prenyl multimerizes neither with itself nor with wild-type (palmitoylated) PSD-95.

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