Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits

Abstract

LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

Figure 1

Effect of LMP7 on processing of LMP2 in transfected T2 cells. Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1 (7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse subunits and cross-react with human subunits. Immunoprecipitates from 5 × 10⁶ cells were loaded per lane. The C3 immunoblot demonstrates the relative amounts of proteasomes in each sample.

Figure 2

Effect of LMP7 active site mutations on LMP2 processing. Proteasomes were immunoprecipitated with MCP21 from lysates of T2 cells transfected with LMP2 and LMP7 (2 + 7), LMP2 and LMP7(T1A) [2 + 7(T1A)], and LMP2 and LMP7(K33A) [2 + 7(K33A)]. Specific subunits were visualized by immunoblotting after SDS-PAGE. Immunoprecipitates from 10⁷ cells were loaded per lane. ppLMP7, partially processed LMP7. Partial loss of the pre-LMP7 band in lane 2 + 7 is due to poor transfer as a result of an air bubble.

Figure 3

Sucrose gradient fractionation of proteasomes from transfected T2 cells. Lysates of 4 × 10⁷ cells were separated on sucrose gradients, and then mature and precursor proteasomes were immunoprecipitated from individual fractions with MCP21. Specific subunits were visualized by immunoblotting after SDS-PAGE, with one-quarter of each immunoprecipitation loaded per lane. Fractions 1–6 out of 11 are shown, with fraction 1 representing the bottom of the gradient.

Figure 4

Relative levels of proteasome subunits in spleen cells from B10 (wild type), LMP7^−/−, and LMP2^−/− mice. Lysates of mouse spleen Con A–stimulated T cell blasts (1.5 × 10⁶/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts of proteasomes and total protein present in each sample.

Figure 5

Pulse-chase labeling of proteasomes synthesized in spleen cells from NIH Black Swiss (wild type), LMP7^−/−, and LMP2^−/− mice. Equal numbers of mouse spleen Con A–stimulated T cell blasts were labeled with [³⁵S]methionine/cysteine for 45 min and then harvested (45 minute pulse) or chased for 8 h (45 minute pulse + 8 hour chase). At the end of each incubation, cells were lysed and proteasomes were immunoprecipitated from half of each lysate with anti-C8, which recognizes only 12-16S preproteasomes (anti-C8), and from the other half of each lysate with anti-C9, which recognizes 20S proteasomes and a small subset of 12-16S preproteasomes (anti-C9) (22). (Anti-C9 immunoprecipitates after a 45-min pulse demonstrated little radioactivity in 20S proteasomes and are not shown.) Proteasome subunits were separated by two dimensional NEPHGE-PAGE and visualized by autoradiography. Spots corresponding to inducible subunits and their precursors, as identified previously (22), are labeled (p2, pre-LMP2; 2, LMP2; p7, pre-LMP7; 7, LMP7; pM, pre-MECL; M, MECL). Note that pre-LMP7 is partially obscured by a larger spot (iota) in NIH Black Swiss preproteasomes. LMP7, LMP2, and their precursors have slightly different isoelectric points in NIH Black Swiss mice due to known polymorphisms (34). Immunoprecipitates from 5 × 10⁶ cells were loaded on each gel.

Figure 6

Cooperative model for proteasome assembly. One major pathway leads to immunoproteasomes containing all three IFN-γ–inducible catalytic β subunits (LMP2, LMP7, and MECL). Another major pathway leads to constitutive proteasomes containing all three constitutive catalytic β subunits (delta [δ], X, and Z). Minor pathways lead to mixed proteasomes containing assortments of both inducible and constitutive catalytic subunits. Appendages represent removable presequences. Subunit positions are based on the structure of the yeast 20S proteasome and assume that inducible subunits occupy the same sites as their constitutive homologues (3).