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

Griffin TA, Nandi D, Cruz M, Fehling HJ, Kaer LV, Monaco JJ, Colbert RA - J. Exp. Med. (1998)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

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.

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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.
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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.

Mentions: Removal of β subunit presequences is associated with maturation of 20S proteasomes (23, 24). Therefore, we hypothesized that pre-LMP2, which accumulates in T2.LMP2 cells, remains in preproteasomes. To test this, cell extracts were fractionated on sucrose gradients and C3-containing proteasomes (both 20S proteasomes and 12-16S preproteasomes) were immunoprecipitated from each fraction and analyzed by immunoblotting for specific subunits (Fig. 3). Indeed, pre-LMP2 was found exclusively in lower mol wt fractions 4 and 5, comigrating with a portion of C3, consistent with α subunit–containing preproteasomes. The small amount of processed LMP2 in these cells (fraction 2) comigrated with other processed β subunits (delta and X), as well as the majority of C3, consistent with 20S proteasomes. These results agree with previous demonstrations of pre-LMP2 in preproteasomes from transfected T2 cells overexpressing mouse LMP2 (23, 32). Of note, there was no detectable pre-delta or pre-X in the pre-LMP2–containing fractions. In contrast, when LMP7 was coexpressed with LMP2 (T2.LMP2/ LMP7), processed LMP2 predominated and comigrated with processed LMP7 and the bulk of C3, consistent with 20S proteasomes (Fig. 3). These results demonstrate that preproteasomes containing pre-LMP2 accumulate in the absence of LMP7, despite the presence of its homologue, X. In contrast, pre-LMP2–containing preproteasomes do not accumulate in the presence of LMP7, suggesting a strong preference for the subsequent incorporation of LMP7 rather than X into these assembling proteasomes. It is important to note that LMP7 can be incorporated into 20S proteasomes in the absence of LMP2 (Fig. 1 and data not shown), suggesting that preproteasomes lacking pre-LMP2 do not have a strong preference for the incorporation of X or LMP7.


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

Griffin TA, Nandi D, Cruz M, Fehling HJ, Kaer LV, Monaco JJ, Colbert RA - J. Exp. Med. (1998)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199179&req=5

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.
Mentions: Removal of β subunit presequences is associated with maturation of 20S proteasomes (23, 24). Therefore, we hypothesized that pre-LMP2, which accumulates in T2.LMP2 cells, remains in preproteasomes. To test this, cell extracts were fractionated on sucrose gradients and C3-containing proteasomes (both 20S proteasomes and 12-16S preproteasomes) were immunoprecipitated from each fraction and analyzed by immunoblotting for specific subunits (Fig. 3). Indeed, pre-LMP2 was found exclusively in lower mol wt fractions 4 and 5, comigrating with a portion of C3, consistent with α subunit–containing preproteasomes. The small amount of processed LMP2 in these cells (fraction 2) comigrated with other processed β subunits (delta and X), as well as the majority of C3, consistent with 20S proteasomes. These results agree with previous demonstrations of pre-LMP2 in preproteasomes from transfected T2 cells overexpressing mouse LMP2 (23, 32). Of note, there was no detectable pre-delta or pre-X in the pre-LMP2–containing fractions. In contrast, when LMP7 was coexpressed with LMP2 (T2.LMP2/ LMP7), processed LMP2 predominated and comigrated with processed LMP7 and the bulk of C3, consistent with 20S proteasomes (Fig. 3). These results demonstrate that preproteasomes containing pre-LMP2 accumulate in the absence of LMP7, despite the presence of its homologue, X. In contrast, pre-LMP2–containing preproteasomes do not accumulate in the presence of LMP7, suggesting a strong preference for the subsequent incorporation of LMP7 rather than X into these assembling proteasomes. It is important to note that LMP7 can be incorporated into 20S proteasomes in the absence of LMP2 (Fig. 1 and data not shown), suggesting that preproteasomes lacking pre-LMP2 do not have a strong preference for the incorporation of X or LMP7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

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.

Show MeSH