Loss of the tubulin-binding protein STMN2 is implicated in amyotrophic lateral sclerosis (ALS), but how it protects neurons is not known. STMN2 is known to turn over rapidly and accumulate at axotomy sites. We confirmed fast turnover of STMN2 in U2OS cells and iPSC-derived neurons and showed that degradation occurs mainly by the ubiquitin-proteasome system. The membrane targeting N-terminal domain of STMN2 promoted fast turnover, whereas its tubulin binding stathmin-like domain (SLD) promoted stabilization. Proximity labeling and imaging showed that STMN2 localizes to trans-Golgi network membranes and that tubulin binding reduces this localization. Pull-down assays showed that tubulin prefers to bind to soluble over membrane-bound STMN2. Our data suggest that STMN2 interconverts between a soluble form that is rapidly degraded unless bound to tubulin and a membrane-bound form that does not bind tubulin. We propose that STMN2 is sequestered and stabilized by tubulin binding, while its neuroprotective function depends on an unknown molecular activity of its membrane-bound form.

Proximity labeling was performed on cells expressing APEX2-fused STMN2 constructs. For STMN2-APEX2 expressing U2OS cells, protein expression was induced for 16 hr by adding 2 μg/ml doxycycline to the growth media. For STMN2-APEX2 expressing neurons, labeling was performed directly on the indicated day. Biotinyl tyramide (Toronto Research Chemicals, B397770) was added to the media at a final concentration of 0.5 μM, and cells were incubated in the labeling media for 1 hr. To initiate labeling, H₂O₂ (Sigma-Aldrich, H1009) was added to a final concentration of 1 mM. Exactly 1 min after H₂O₂ treatment, the labeling solution was decanted, and cells were washed three times with cold quenching solution containing 10 mM sodium ascorbate (VWR 95035-692), 5 mM Trolox (Sigma-Aldrich, 238813), and 10 mM sodium azide (Sigma-Aldrich, S2002). The cell lysate was harvested by scraping and cleared by centrifugation at 16,000 × g for 20 minutes, and the resulting pellet was flash-frozen and stored at −80°C until streptavidin pull-down. The cell pellet was lysed using a cell lysis solution (8 M Urea, 100 mM sodium phosphate, pH 8, 1% SDS (w/v), 100 mM NH4HCO3, 10 mM TCEP, sterile-filtered). Protein was extracted by adding 55% TCA (Sigma-Aldrich, 91228) at a 1:1 ratio to the lysate and precipitated by centrifugation. The protein pellet was then washed three times with −20°C cold acetone. Protein was subjected to cysteine alkylation using 20 mM iodoacetamide (Sigma-Aldrich, I6125), quenched by 50 mM DTT (Sigma-Aldrich, 43815), and pulled down using streptavidin magnetic beads (VWR, PI88817). A detailed procedure for the protein processing procedure can be found in Kalocsay, 2019.

Subsequent protein processing procedures and MS analysis were carried out as described. The digested peptides were labeled with TMTpro 16-plex (Thermo Fisher Scientific, A44520) for 1 h. Data collection followed a MultiNotch MS3 TMT method using an Orbitrap Lumos mass spectrometer coupled to a Proxeon EASY-nLC 1200 liquid chromatography system (both Thermo Fisher Scientific). Peptides were searched against a size-sorted forward and reverse database of the Homo sapiens reference proteome (Uniprot 03/2021) using SEQUEST (v.28, rev. 12)-based software. Spectra were first converted to mzXML. For the searches, a mass tolerance of 20 p.p.m. for precursors and a fragment ion tolerance of 0.9 Da were used. The search allowed for a maximum of two missed cleavages per peptide.

Carboxyamidomethylation on cysteine was set as a static modification (+57.0214 Da), and oxidized methionine residues (+15.9949 Da) were searched for dynamically. A target decoy database strategy was applied, and a false discovery rate (FDR) of 1% was set for peptide-spectrum matches after filtering by linear discriminant analysis. The FDR for final collapsed proteins was 1%. MS1 data were calibrated post-search, and searches performed again. Quantitative information on peptides was derived from MS3 scans. Quantitative tables were generated requiring an MS2 isolation specificity of >70% for each peptide and a sum of TMT (tandem mass tags) signal:noise ratio (s:n) of >200 over all channels for any given peptide, and then exported to Excel and further processed therein. Proteomics raw data and search results were deposited in the PRIDE archive. The relative summed TMT s:n for proteins between two experimental conditions was calculated from the sum of TMT s:n for all peptides of a given protein quantified.