Abstract
Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.
Highlights
signal peptide peptidase (SPP) was discovered as an intramembrane-cleaving proteases (ICLiPs) responsible for removing hydrophobic signal peptides liberated from the N terminus of secreted or transmembrane proteins by signal peptidase (SP) cleavage during their co-translocation into the endoplasmic reticulum (1)
Differential Shedding of Bri2 and Bri3— substrate requirements for ␥-secretase (Ref. 26; summarized in Refs. 17 and 33) and SPP (29) are at least partially described, very little is known about determinants that allow SPPLs to recognize their appropriate protein substrates
We searched for substrate requirements of SPPL2b, an ICLiP of the GXGD-type located within the late secretory compartments including plasma membrane and endosomes, because this may allow a direct comparison of the properties of ␥-secretase with those of the SPPLs
Summary
Cell Culture, cDNAs, and Transfection—HEK-293EBNA (HEK-293) were cultured in Dulbecco’s modified Eagle’s medium with Glutamax (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). HEK-293 single cell clones stably expressing SPPL2b or SPPL2b D421A containing a C-terminal hemagglutinin tag (AYPYDVPDYA) have been described before (24). CDNAs encoding chimeric proteins (Table 1) were generated by PCR. Transient transfection of HEK293 cell lines stably expressing the indicated SPPL2b variants was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Antibodies, Immunoprecipitation, and Immunoblotting— The monoclonal anti-FLAG M2 and the polyclonal hemagglutinin 6908 antibody were obtained from Sigma; the poly- and monoclonal V5 antibodies were purchased from Chemicon (Schwalbach, Germany) and Invitrogen, respectively. The polyclonal antibodies against ADAM-10 and calnexin were purchased from Calbiochem and Stressgene/Biomol (Hamburg, Germany). The monoclonal anti-Giantin antibody (ALX-804 – 600) was obtained from Alexa (AXXORA Deutschland GmbH, Lorach, Germany). 22C11 antibody recognizing the N terminus of APP was purchased from Chemicon (Schwalbach, Germany). Amino acid identity and similarity were calculated using EMBOSS pairwise alignment algorithms
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