Proteasomes play a major role in non-lysosomal proteolysis and also in the 
processing of proteins for presentation by the MHC class I pathway. In animal 
cells they exist in several distinct molecular forms which contribute to the 
different functions. 26S proteasomes contain the core 20S proteasome together 
with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the 
ends of the 20S proteasome to form PA28-proteasome complexes and 
PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome 
subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We 
have found differences in the subcellular distribution of the different forms of 
proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are 
predominantly located in the cytoplasm, while 19S regulatory complexes (present 
at significant levels only in 26S complexes) are present in the nucleus as well 
as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic 
reticulum (ER) where they may facilitate the generation of peptides for 
transport into the lumen of the ER. We have also investigated the effects of 
gamma-interferon on the levels and subcellular distribution of inducible 
subunits and regulator subunits. In each case gamma-interferon was found to 
increase the level but not to alter the distribution. Several subunits of 
proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 
(alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that 
gamma-interferon treatment decreases the level of phosphorylation of 
proteasomes. We have investigated the role of phosphorylation of C8 by casein 
kinase II by site directed mutagenesis. The results demonstrate that 
phosphorylation at either one of the two sites is essential for the association 
of 19S regulatory complexes and that the ability to undergo phosphorylation at 
both sites gives the most efficient incorporation of C8 into the 26S proteasome.