DOI: 10.5061/dryad.vhhmgqp51
title: Tubulin regulates the stability and localization of STMN2 by binding to its soluble form

description: 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.
description: 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, H<sub>2</sub>O<sub>2</sub>
 (Sigma-Aldrich, H1009) was added to a final concentration of 1 mM. Exactly
 1 min after H<sub>2</sub>O<sub>2</sub> 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 <em>Homo
 sapiens</em> 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 &gt;70% for each peptide and a sum of TMT (tandem mass tags)
 signal:noise ratio (s:n) of &gt;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.
description: # Tubulin regulates the stability and localization of STMN2 by binding to
 its soluble form
 [https://doi.org/10.5061/dryad.vhhmgqp51](https://doi.org/10.5061/dryad.vhhmgqp51) ## Description of the data and file structure * D3iPSCNeuron_STMN2APEX_GO_Cellular_Component_2017.csv\ Gene Ontology analysis of proximity proteomics data collected from Day 3 neurons by GO Cellular Component 2017. * D3iPSCNeuron_STMN2APEX_ProximityProteomics_NormalizationFactors.csv\ Normalization factors used to normalize raw proximity proteomics data collected from Day 3 neurons. * D3iPSCNeuron_STMN2APEX_ProximityProteomics.csv\ Proximity proteomics data collected from Day 3 neurons. * U2OS_STMN2APEX_GO_Cellular_Component_2017.csv\ Gene Ontology analysis of proximity proteomics data collected from U2OS cells by GO Cellular Component 2017. * U2OS_STMN2APEX_ProximityProteomics.csv\ Proximity proteomics data collected from U2OS cells. * U2OS_STMN2APEX_ProximityProteomics_NormalizationFactors.csv\ Normalization factors are used to normalize raw proximity proteomics data collected from U2OS cells.