cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome¹

Abstract

We employed cDNA cloning to deduce the complete primary structures of p28 and p40.5, two novel subunits of PA700 (also called 19S complex), a 700 kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consisted of 226 and 376 amino acids with calculated molecular masses of 24 428 Da and 42 945 Da, and isoelectric points of 5.68 and 5.46, respectively. Intriguingly, p28 contained five conserved motifs known as `ankyrin repeats', implying that this subunit may contribute to interaction of the 26S proteasome with other protein(s). Computer-assisted homology analysis revealed high sequence similarities of p28 and p40.5 with yeast proteins, termed Nas6p and Nas7p (non-ATPase subunits 6 and 7), respectively, whose functions are as yet unknown. Disruption of these yeast genes, NAS6 and NAS7, had no effect on cell viability, indicating that neither of the two subunits is essential for proliferation of yeast cells. However, the NAS7, but not NAS6, disruptant cells caused high sensitivity to heat stress, being unable to proliferate at 37°C.

Introduction

Ubiquitin (Ub) and the proteasome are principal components of a major proteolytic system that is responsible for degrading a wide variety of intracellular proteins, including constitutively unstable regulatory proteins and proteins with aberrant structures generated by mutations or various environmental stresses (for details, see reviews by Coux et al., 1996; Hochstrasser, 1996and references therein). These and other cellular proteins are targeted for degradation by the 26S proteasome after they have been covalently attached to Ub in the form of a poly-Ub chain. The 26S proteasome is composed of two large multi-protein complexes: the 20S proteasome and PA700 (also known as 19S complex). The 20S proteasome is a protease with a molecular mass of 700 kDa. It is a cylinder-shaped particle formed by the axial stacking of four heptameric rings (Baumeister et al., 1998). In eukaryotes, the two outer rings have identical compositions of seven distinct but homologous subunits, termed α. Likewise, the two inner rings each contains seven distinct but homologous subunits, termed β. The crystal structure of the yeast 20S proteasome, together with related biochemical and molecular studies, indicate that three β-type subunits of each inner ring serve as catalytic sites (Groll et al., 1997). These sites face the interior of the cylinder and reside in a chamber formed by the centers of the abutting β rings. The crystal structures of yeast and archaebacterial proteasomes suggest that substrates gain access to the active sites only after passing through a narrow (13 Å) opening corresponding to the center of the α rings (Lõwe et al., 1995; Groll et al., 1997). The yeast structure shows that the amino-termini of the α subunits form an additional physical barrier for substrates to reach the active sites.

The 20S proteasome forms the proteolytic core of the 26S proteasome, but does not display functional or regulatory properties of the 26S proteasome. These characteristics are conferred upon the proteasome by PA700. PA700 is a 700 kDa protein complex composed of about 20 subunits (Ma et al., 1994; DeMartino et al., 1994). These subunits range in molecular mass from 25  to 110 kDa and represent distinct gene products. PA700 binds to both of the proteasomes' α rings to form the active 2000 kDa complex or the 26S proteasome. The 26S proteasome, unlike the 20S proteasome, degrades ubiquitinylated proteins in an ATP-dependent fashion. Although the molecular mechanism of protein degradation by the 26S proteasome is unknown, PA700 may regulate and promote the translocation of polypeptide substrates through the proteasome's terminal rings to the active sites.

In order to understand the molecular basis of the function and regulation of the 26S proteasome, we and others have been determining the primary structures of individual subunits of PA700 (for review, see Tanaka, 1998). Structures for many of these subunits have now been established. For example, six subunits are distinct members of a large protein family characterized by a 200 amino acid-domain containing ATP-binding motifs (Beyer, 1997). One or more of these subunits presumably participates in the ATP-dependent assembly of the 26S proteasome and in the ATP-dependent degradation of ubiquitinylated proteins. Primary structures for many of the non-ATPase subunits have also been determined and have been shown to be unrelated to one another (Tanaka and Tsurumi, 1997). These structures, however, have not helped to define the biochemical functions of the respective proteins. Additional biochemical studies have indicated roles for at least two non-ATPase subunits of PA700. One subunit, S5a (Sun1p, Mcb1 in yeast), binds poly-Ub chains, which may determine specificity of the 26S proteasome for ubiquitinylated proteins (Ferrell et al., 1996). Another subunit, p37, appears to be an isopeptidase that disassembles the poly-Ub chain (Lam et al., 1997).

The current work reports the primary structures of two additional subunits, termed p28 and p40.5, of the human 26S proteasome. In addition, the homologs of these proteins in yeast, Nas6p and Nas7p, have been identified. We show that these subunits are novel proteins and are not necessary for proliferation in yeast. However, Nas7p is required for viability of yeast at high temperature. Subunit p28 contains five tandemly-repeated segments referred to as ankyrin repeats, which are found in a variety of functionally unrelated proteins and have been implicated in protein–protein interactions (Thompson et al., 1991; Blank et al., 1992). Based on the current results and previous data, the functional properties of the newly defined PA700 subunits in proteasome-mediated proteolysis are discussed.