Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3.
Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.
Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, Schillerstrasse 44, 80336 Munich, Germany.
Peter
P
Krawitz
Christof
C
Haffner
Regina
R
Fluhrer
Harald
H
Steiner
Bettina
B
Schmid
Christian
C
Haass
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001224:Aspartic Acid / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D018390:Gene Targeting; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D053764:Presenilin-1; D021382:Protein Sorting Signals; D016372:RNA, Antisense / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D021382:Protein Sorting Signals; D016372:RNA, Antisense; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D001224:Aspartic Acid; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; C513920:sppl2 protein, zebrafish