Drosophila proteasome Dm25 subunit substitutes the mouse MC3 subunit in hybrid proteasomes. The N-terminal domain is essential for subunit incorporation.

A Seelig,G Multhaup,B Pesold-Hurt,K Beyreuther,P M Kloetzel

doi:10.1016/s0021-9258(19)74428-7

Abstract

The proteasome is a multisubunit 20 S proteinase complex involved in ubiquitin-dependent and -independent intracellular protein metabolism. Individual subunits of the alpha- and beta-type share extensive sequence homology and are encoded as members of two related and evolutionarily conserved gene families. Due to the lack of viable deletion mutants of essential alpha-type proteasome subunits in higher eukaryotes, an identification and analysis of potentially homologous subunits of different species was so far not possible. It is shown here that the novel Drosophila alpha-type Dm25 subunit can be incorporated into mouse proteasomes of stably transfected NIH 3T3 cells. The Dm25 subunit is able to substitute the mouse MC3 alpha-type subunit in proteasomes, indicating a high structural and possibly also functional homology of the two subunits. In contrast and pointing at the importance of the slightly hydrophobic N-terminal region stabile expression of a Dm25 subunit, which is truncated at its N terminus and lacks PROS box I, results in a subunit which cannot be incorporated into mouse proteasomes. The ability to form hybrid proteasomes involving essential nondeletable subunits now opens the possibility for structural and also functional analysis of such subunits by mutagenesis in higher eukaryotes.

Highlights

From the Center of Molecular Bwlogy, University of Heidelberg, Im Neuenheimer Feld 282, 69 120 Heidelberg, Federal Republicof Germany
In the present paper we report Grant SFB 229 K1/C4 and by the Bundesminisbrium f i r Forschung on theidentification and characterizationof a new Drosophila und Technologie.The costs of publication of this article were defrayed proteasome subunit, Dm25
Theidentity of the isolated cDNA clone is demonstrated by comparison of the deduced amino acidsequencewith thepartial peptide sequenceswhichrevealsa 100% amino acid identity in four peptides covering the N-terminal as well as the C-terminal region of the protein

Summary

Oligonucleotideswith restriction sites were used for PCR in order to

Amino Acid Sequencing of Tryptic Peptides-Tryptic peptides of clone the resulting fragments inframe with the N-terminal6histidine the 25-kDa Drosophilaproteasome subunit were separated by reverse residues of the vector. Triton X-100 in PBS for 10 min at room temperature. Affinitypurified antibodies were diluted 1:1, and thecells were incubated for 1-3 h in a moist chamber at room temperature. After incubation cells 361 were washed for 2h with several changes in PBS. The second fluorescein-conjugated antibody (goat anti-rabbit IgG, Dianova) was diluted 1:200in newborn calf serum, and thecells were incubated for. Dm25 subunits 661 containing filter strips were incubated in crude N19 antiserum, and monospecific antibodies were eluted in buffer containing 0.2 M HC1, pH 2.1. Analysis of Protensomes-Mouse proteasomes of NIH 3T3 fibroblast cells were disrupted and extracted in extraction buffer

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RESULTS

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