Purification and characterization of hen oviduct microsomal signal peptidase

Rk, Baker; Mo, Lively

doi:10.1021/bi00400a010

Cited by 80 publications

(28 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since Spc3p and Sec11p function directly in the signal peptide cleavage reaction, it seems likely that the SPC subunits predominately exposed to the lumen comprise a two-subunit core enzyme that is required for signal peptidase activity. This conclusion is supported by the fact that a two-subunit avian signal peptidase containing homologs to Spc3p and Sec11p has been shown to function in the signal peptide cleavage reaction in vitro (16).…”

Section: Discussionmentioning

confidence: 92%

In Addition to SEC11, a Newly Identified Gene,SPC3, Is Essential for Signal Peptidase Activity in the Yeast Endoplasmic Reticulum

Fang

Mullins

Green

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Among the three characterized subunits comprising the signal peptidase complex of the yeast Saccharomyces cerevisiae (Sec11p, Spc1p, and Spc2p), only Sec11p is essential for cell growth, signal peptide cleavage, and signal peptidase-dependent protein degradation. Here we report the cloning of the SPC3 gene encoding the homolog to mammalian signal peptidase subunit SPC22/ 23. We find that Spc3p is also required for cell growth and signal peptidase activity within the yeast endoplasmic reticulum.Amino-terminal signal sequences of proteins targeted to the endoplasmic reticulum (ER) 1 are cleaved by a membranebound endoprotease termed signal peptidase (1, 2). This enzyme has also been shown to catalyze the proteolytic fragmentation of some abnormal membrane proteins, thereby leading to their degradation (3, 4). Isolated from the yeast Saccharomyces cerevisiae, ER signal peptidase (SP) contains four nonidentical protein subunits (5-7). Three of the subunits of this signal peptidase complex (SPC) have been functionally examined. Sec11p (17 kDa) is required for signal peptide cleavage (8) and signal peptidase-dependent protein degradation (4). Sec11p is related to two subunits of the mammalian SPC (SPC18 and SPC21) (9, 10). In contrast to Sec11p, the Spc1p (11 kDa) and Spc2p (18 kDa) subunits of the yeast SPC are nonessential for cell growth and enzyme activity (6, 7). Spc1p and Spc2p, however, perform auxiliary and nonredundant roles. Spc1p facilitates signal peptide cleavages in cells burdened with high levels of a membrane protein substrate of the signal peptidase-dependent protein degradation pathway (6). Spc2p is important for enzyme activity and cell viability at elevated temperatures (7). Spc1p and Spc2p are homologous to mammalian subunits SPC12 and SPC25, respectively (6,7,11,12).Despite the fact that a multisubunit signal peptidase has been purified from yeast and mammalian cells, enzymes exhibiting less subunit complexity have been identified in other systems. Leader peptidase from the inner membrane of E. coli consists of a single polypeptide chain (13). This protein exhibits limited homology to Sec11p and to mammalian SPC18 and SPC21 (14, 15). Signal peptidase purified from hen oviduct contains two subunits, one related to Sec11p and a second related to SPC22/23 of the mammalian SPC (16 -19).In the present study, we have cloned and characterized the SPC3 gene encoding the yeast homolog to mammalian SPC22/ 23. We find that, as with Sec11p, Spc3p is essential for signal peptide cleavage and signal peptidase-dependent protein degradation. Our data are thus in agreement with in vitro studies, using an avian system, which show that a two-subunit complex functions to cleave signal peptides. The idea that both Sec11p and Spc3p are related to the prokaryotic signal peptidase is also discussed. EXPERIMENTAL PROCEDURESPlasmid Constructs Bearing the SPC3 Gene-Plasmid pSPC3 bearing SPC3 was isolated from a high copy (2 m) plasmid library marked with LEU2 (American Type Culture Collection ATCC No. 37323). Among 5,500 ...

show abstract

Section: Discussionmentioning

confidence: 92%

In Addition to SEC11, a Newly Identified Gene,SPC3, Is Essential for Signal Peptidase Activity in the Yeast Endoplasmic Reticulum

Fang

Mullins

Green

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Therefore, it has been speculated, that SPC12 and SPC25 are involved in processes other than substrate binding or the actual enzymatic reaction. This assumption was supported by the fact that an active signal peptidase that comprises only an SPC18 homolog and an SPC22/23 homolog could be purified from hen oviduct (19,20).…”

Section: From the Max-delbrü Ck-center For Molecular Medicine Robertmentioning

confidence: 95%

“…This notion has been supported by two other facts. 1) Enzymatically active signal peptidase may be purified from hen oviduct as a complex of only two subunits (SPC18 and gp23) (19), and 2) neither the SPC12 homolog, Spc1p, nor the SPC25 homolog, Spc2p, is essential for viability or signal peptidase activity in S. cerevisiae. The identification of Spc3p as a second essential component of yeast SPC described above demonstrates that there must be indeed a substantial difference in the mode of signal peptide cleavage in eucaryotes and procaryotes.…”

Section: Figmentioning

confidence: 99%

The Yeast SPC22/23 Homolog Spc3p Is Essential for Signal Peptidase Activity

Meyer

Hartmann

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

In eucaryotic cells signal sequences of secretory and membrane proteins are cleaved by the signal peptidase complex during their transport into the lumen of the endoplasmic reticulum. The signal peptidase complex in yeast consists of four subunits. To date, three of these subunits have been functionally characterized. One of them, the Sec11p, is essential for viability of yeast cells. It shows significant homology to the mammalian SPC18 and SPC21 as well as to bacterial leader peptidases. Two other subunits, Spc1p and Spc2p, have been shown to be homologous to mammalian SPC12 and SPC25, respectively, and are not essential for protein translocation or signal peptide cleavage.We have purified and analyzed the fourth subunit of yeast signal peptidase, Spc3p. The protein is essential for viability of yeast cells. Depletion of SPC3 leads to accumulation of precursors of secretory proteins in vivo and to the loss of the signal peptidase activity in vitro. Therefore, in contrast to the bacterial leader peptidases, yeast signal peptidase requires a second subunit for its function.

show abstract

“…SPC18 and SPC21 have high identity to each other [11] and are homologous to Sec11 [6], [12], [13]. SPC22/23 is homologous to Spc3p [14]–[17] while SPC12 and SPC25 are homologous to Spc1p and Spc2p, respectively [5], [9], [18], [19] (Table 1). SPC18, SPC21 and SPC22/23 are single-pass transmembrane proteins, the bulk of which reside within the ER lumen.…”

Section: Introductionmentioning

confidence: 99%

Drosophila Signal Peptidase Complex Member Spase12 Is Required for Development and Cell Differentiation

et al. 2013

View full text Add to dashboard Cite

It is estimated that half of all proteins expressed in eukaryotic cells are transferred across or into at least one cellular membrane to reach their functional location. Protein translocation into the endoplasmic reticulum (ER) is critical to the subsequent localization of secretory and transmembrane proteins. A vital component of the translocation machinery is the signal peptidase complex (SPC) - which is conserved from yeast to mammals – and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Failure to cleave the SP, due to mutations that abolish the cleavage site or reduce SPC function, leads to the accumulation of uncleaved proteins in the ER that cannot be properly localized resulting in a wide range of defects depending on the protein(s) affected. Despite the obvious importance of the SPC, in vivo studies investigating its function in a multicellular organism have not been reported. The Drosophila SPC comprises four proteins: Spase18/21, Spase22/23, Spase25 and Spase12. Spc1p, the S. cerevisiae homolog of Spase12, is not required for SPC function or viability; Drosophila spase12 null alleles, however, are embryonic lethal. The data presented herein show that spase12 LOF clones disrupt development of all tissues tested including the eye, wing, leg, and antenna. In the eye, spase12 LOF clones result in a disorganized eye, defective cell differentiation, ectopic interommatidial bristles, and variations in support cell size, shape, number, and distribution. In addition, spase12 mosaic tissue is susceptible to melanotic mass formation suggesting that spase12 LOF activates immune response pathways. Together these data demonstrate that spase12 is an essential gene in Drosophila where it functions to mediate cell differentiation and development. This work represents the first reported in vivo analysis of a SPC component in a multicellular organism.

show abstract

Purification and characterization of hen oviduct microsomal signal peptidase

Cited by 80 publications

References 39 publications

In Addition to SEC11, a Newly Identified Gene,SPC3, Is Essential for Signal Peptidase Activity in the Yeast Endoplasmic Reticulum

In Addition to SEC11, a Newly Identified Gene,SPC3, Is Essential for Signal Peptidase Activity in the Yeast Endoplasmic Reticulum

The Yeast SPC22/23 Homolog Spc3p Is Essential for Signal Peptidase Activity

Drosophila Signal Peptidase Complex Member Spase12 Is Required for Development and Cell Differentiation

Contact Info

Product

Resources

About