Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.Cleavable signal sequences, which are usually located at the N termini of precursor proteins, function in the delivery of their protein cargo to specific destinations within both eukaryotic and eubacterial cells. A large number of signal sequences possess a common motif consisting of an n-region that is often positively charged and a hydrophobic core (the h-region) followed by a polar c-region containing the cleavage site (1). Small uncharged amino acids are usually present at the Ϫ1 and Ϫ3 positions from the cleavage site. Signals exhibiting this motif are recognized and cleaved by type I signal peptidases. Type I signal peptidases are found within the endoplasmic reticulum (ER) 1 membrane, the mitochondrial inner membrane, and the cytoplasmic membrane of eubacterial cells (reviewed in Ref. 2). There is a strong functional conservation of the signal sequence cleavage reaction, revealed by the fact that signal sequences of proteins targeted normally to the ER can be cleaved by eubacterial signal peptidase (3), and eubacterial signal sequences can be cleaved by ER signal peptidase (4).Site-directed mutagenesis studies suggest that many eubacterial signal peptidases contain a serine/lysine dyad with which to catalyze the cleavage reaction (5-9). A similar catalytic dyad is thought to be present in the LexA and UmuD proteins of Escherichia coli (10, 11) and in both catalytic subunits of mitochondrial signal peptidase (12). Recent x-ray crystallographic analysis confirms the role of serine as a nucleophile and is consistent with a lysine acting as a general base in catalysis by E. coli leader peptidase (13).At least one eubacterial signal peptidase, SipW of Bacillus subtilus, may exhibit a catalytic site more like that of Sec11p of the ER signal peptidase. B. subtilus contains five distinct chromosomally encoded signal peptidases, SipW being the only one like Sec11p (14, 15). As shown in Fig. 1, Sec11p contains a serine residue that aligns to the catalytic serine of leader peptidase; however, within the limited regions of homology that exist between Sec11p and leader peptidase, Sec11p contains a histidine that has been aligned to the catalytic lysine of leader peptidase (16,17). From this, the type I signal peptidase family may contain a subgroup, represented by Sec11p and SipW, that uses a distinct catalytic mechanism (14). It has been noted previously, however, that an alignment of Sec11p to the E. col...

No abstract

Signal peptidase removes amino-terminal signal peptides from precursor proteins during or immediately following their translocation to the lumen of the endoplasmic reticulum (ER) and may participate in ER degradation, a poorly defined process whereby abnormal proteins are rapidly degraded early in the secretory pathway. Here, the involvement of signal peptidase in ER degradation is examined through the use of two chimeric membrane proteins that lack amino-terminal signal peptides: A189invHD, which contains sequences derived from arginine permease and histidinol dehydrogenase, and AHDK2, containing the ER-resident protein Kar2p fused to the carboxyl terminus of A189invHD. Degradation of approximately 95% of A189invHD is observed in yeast cells expressing enzymatically active signal peptidase, whereas only 60% undergoes rapid degradation in a sec11 mutant bearing a temperature-sensitive mutation in the gene encoding the 18-kDa subunit (Sec11p) of the signal peptidase complex. AHDK2 is proteolyzed in a reaction yielding at least two fragments in wild-type cells and in the sec11 mutant containing a plasmid bearing the SEC11 gene. The proteolytic reaction is catalyzed in a temperature-dependent manner in the sec11 mutant, with AHDK2 remaining stable at the nonpermissive temperature. Using conditional mutants defective in protein translocation into and out of the ER and in vitro protease protection studies, the site of degradation for AHDK2 is localized to the ER lumen. The data therefore indicate (i) A189invHD is degraded through both signal peptidase-dependent and independent processes; (ii) signal peptidase, specifically the Sec11p subunit, is required for the proteolysis of AHDK2; and (iii) the Kar2 fragment at the carboxyl terminus of AHDK2 permits detection of proteolytic intermediates.

Two subunits of the mammalian signal peptidase complex, SPC12 and SPC25, share similar membrane topologies with the majority of each protein oriented toward the cytoplasm. Such similarities may suggest that these proteins perform redundant functions in signal peptidase activity. In the present study, we addressed this issue through analysis of the yeast homologs to SPC12 and SPC25, Spc1p and Spc2p. We show that both Spc1p and Spc2p are nonessential for signal peptidase activity and growth of yeast cells and that null mutations in the genes encoding Spc1p and Spc2p are synthetically lethal with a conditional mutation affecting Sec11p, an essential subunit of yeast signal peptidase. However, a high copy plasmid encoding Spc1p suppresses the conditional sec11 mutation, whereas the corresponding plasmid encoding Spc2p does not suppress sec11. Moreover, Spc2p, but not Spc1p, is important for signal peptidase activity and cell viability at high temperatures. These results indicate that although both Spc1p and Spc2p are noncatalytic, they are functionally distinct. Evidence is also presented that a double mutant lacking Spc1p and Spc2p grows well relative to wild type yeast cells, indicating that the signal peptidase complex missing at least two of its subunits is sufficient for signal peptidase activity in vivo.