Fig. 1.

NMPs are present in tadpole neurons. (A) Biochemical evidence for NMPs in the tadpole brain. Tadpole brain tissue was collected at 30 m or 6 h after either BE or Epox injection, and an equivalent amount of protein from the membrane and cytosolic fractions were labeled with indicated antibodies on western blots. The 20S core proteasome subunits (α1–7) are abundantly expressed in the tadpole brain and are enriched in the membrane fraction. Importantly, the biotin signal was recovered at the molecular weight corresponding to the 20S catalytic subunits (run around 20 to 25 kD) bound to the injected BE in (and only in) the membrane preparation both at 30 min and 6 h after the injection. Actin, a cytosolic protein, and GluA1, which is enriched in the neuronal membrane, serve as positive controls for the cell fractionation preparation. (B) Preinjection of Epox occluded BE binding to NMPs. Brain lysates from uninjected, BE-injected, and Epox preinjected (Epox-BE-inj) samples were purified by neutravidin pulldown to coprecipitate BE-bound proteins and blotted with biotin and 20S β5 antibody. (C) Anatomical illustration of cellular organization of the optic tectum (OT) and the ventricle (V). CBL: neuronal cell body layer. NPL: neural progenitor cell layer. N: neuropil. (D) Immunohistological localization of NMPs bound to BE. Vibratome sections of the OT immunostained with Anti-Biotin antibody show punctate biotin signal in neuronal cell body layers and the neuropil in the BE-injected tectum but not uninjected, or Epox-preinjected, or biotin-injected controls. All samples processed in parallel under the same conditions. Gold: biotin; blue: DAPI. (Scale bar, 20 µm.) (E–J) Ultrastructural distribution of the 20S proteasome shown by preembedding immunogold labeling with anti-α1-7 20S proteasome subunit antibody and FluoroNanogold secondary antibody (1.4-nm-diameter gold particles). Plasma membranes are highlighted in light blue. (E and F) Ultramicrographs from the tectal cell body layer showing anti-α1-7 20S immunogold labeling (E) and no primary controls (F). Boxed regions are shown at higher magnification in E’ and F’. Immunogold particles identifying 20S α subunits are associated with the plasma and nuclear membranes and found in the cytoplasm. Asterisks mark nuclei. (Scale bar, 1 µm.) (G–J) Ultramicrographs from the tectal neuropil labeled with anti-α1-7 20S antibodies (G and I) or no primary control (H and J). Numerous immunogold particles identifying 20S α subunits are localized at or near the plasma membrane in G and I. The 20S α subunits are also visible at synaptic sites, which are marked by arrows. (Scale bar, 500 nm.)