The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins

Abstract

Signal peptide peptidase (SPP) and SPP‐like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II‐oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER‐resident family members SPP and SPPL2c were shown to also cleave tail‐anchored proteins, including selected SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co‐expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail‐anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1‐4 levels in tissues and primary cells of SPPL2a/b double‐deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1‐4 in a cell type‐ and tissue‐dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1‐4 by initiating the degradation of these proteins, which might impact cellular trafficking.

Unravelling the complexity of enzyme catalysis

“…In their Commentary article [10], Kv eta Tr avn ı ckov a and Kvido St r ı sovsk y discuss the work of Moritz Ballin et al [11] (also collected in this Focus Issue), which implicates SPPL2a/b intramembrane proteases in the cleavage of SNARE proteins and thereby regulation of SNARE protein turnover. In a second Commentary article, on keratinocytes and skin diseases, Jenna Demeter and colleagues comment on the work of Simon Nold et al (also included in this Focus Issue), referring to the potential of pyruvate kinase muscle isoforms as therapeutic targets [12,13].…”

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling

“…Tail‐anchored (TA) proteins are a diverse class of membrane proteins that are post‐translationally inserted with their C terminus into membranes by a specialized machinery [49]. Initially, only SPP and SPPL2c had been shown to cleave type IV proteins [50], but very recently the type IV SNARE proteins VAMP1‐4 have been added to the list of SPPL2a and SPPL2b substrates [51]. All four VAMPs are so‐called R‐SNAREs with a smaller cytosolic domain than Q‐SNAREs.…”

“…Their larger N-terminal domain co-localizes with the cytosolic C-terminal domains of the SPP/SPPL proteases. In vitro experiments point to a size limitation of the cytosolic domain of cleavable type IV proteins [51,52]. In addition, a few polytopic type III membrane proteins have been found to be substrates to cleavage by SPP/SPPL proteases (Table 1).…”

On the track of intramembrane clippers: the SPPL2a/b proteases caught in the act in animal models

“…The main findings of Ballin et al. [13]: SPPL2a/b proteases cleave the tail‐anchored proteins R‐SNAREs (smaller cytosolic domains) VAMP1, 2, 3 and 4 but spare the tail‐anchored proteins Q‐SNAREs (large cytosolic domains) and some other R‐SNAREs. These novel substrates do not require preprocessing before their intramembrane cleavage by SPPL2a/b.…”

“…The article discussed here [13] asks whether SPPL2a/ b proteases can, like SPPL2c, cleave a subset of SNARE proteins and hence contribute to the regulation of their turnover. To answer this question, Ballin et al first employed an overexpression assay and candidate screening.…”