The intramembrane protease SPPL 2c promotes male germ cell development by cleaving phospholamban

Abstract

Signal peptide peptidase (SPP) and the four homologous SPP‐like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II‐oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non‐expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail‐anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c−/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA2)‐regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c−/− mice.

Signal peptide peptidase‐like 2c impairs vesicular transport and cleaves SNARE proteins

“…Confirming the data obtained by proteomics (Fig A and Ref. ), a significant accumulation of Syntaxin 8 was observed in SPPL2c −/− mouse testis (Fig C and D), while Syntaxin 8 mRNA remained unchanged (Fig E), suggesting a lack of Syntaxin 8 processing in SPPL2c −/− mouse testis. However, we were not able to demonstrate the accumulation of other SNARE proteins, most likely because SPPL2c expression in testis was found predominantly in maturating spermatids .…”

“…Expression analysis on mRNA and protein level revealed physiological SPPL2c expression in murine and human testis but so far not in any other tested tissue (Fig A and B, and accompanying manuscript by Niemeyer et al ). Proteomic analysis of SPPL2c −/− testis samples identified Syntaxin 8 as a candidate SPPL2c substrate also in vivo , suggesting cleavage of SNARE proteins by SPPL2c also in vivo . Since expression of SPPL2c has a pronounced effect on vesicular transport, it is comprehensible why it is expressed in hardly any cell or tissue under normal conditions.…”

“…In this study, we identified the first substrates for SPPL2c, a so far orphan protease, using systematic proteomic approaches. While analysis of testis tissue from SPPL2c −/− mice revealed significant enrichment of only a few proteins , ectopic expression of SPPL2c in HEK293 cells resulted in changes in a variety of proteins that mainly cluster to pathways involved in vesicular trafficking, specifically membrane fusion, and protein glycosylation. Given the restricted expression of SPPL2c to maturating spermatids , it is likely that many candidate substrates have been missed in the analysis of whole testis preparations due to dilution effects.…”

“…While analysis of testis tissue from SPPL2c −/− mice revealed significant enrichment of only a few proteins , ectopic expression of SPPL2c in HEK293 cells resulted in changes in a variety of proteins that mainly cluster to pathways involved in vesicular trafficking, specifically membrane fusion, and protein glycosylation. Given the restricted expression of SPPL2c to maturating spermatids , it is likely that many candidate substrates have been missed in the analysis of whole testis preparations due to dilution effects. Similar to proteome‐wide screens on SPPL3 −/− cells and cells with ectopic SPPL3 expression , substrate spectra of SPPL2c overlapped to some extent in the knock out model and the overexpression model, but the majority of candidate substrates differed.…”

“…Besides candidates that were validated as SPPL2c substrates, also protein levels of validated SPP substrates, such as Cyb5a, which were not processed by SPPL2c , were reduced in SPPL2c‐expressing cells (Fig ). Since SPP levels were unchanged in SPPL2c‐expressing cells compared to control cells (Fig EV1) and SPPL2c expression results in ER retention of certain secretory proteins (Fig ), we conclude that SPP likely has more access to some of its substrates due to increased co‐localization, and thus, elevated processing is observed.…”