DOI: 10.5061/dryad.j9kd51crx
title: Tubulin regulates stability and localization of STMN2 by binding preferentially to its soluble form

description: The small, tubulin-binding protein STMN2 is highly expressed in neurons
 and is implicated in amyotrophic lateral sclerosis. STMN2 degrades rapidly
 and accumulates at axotomy sites, suggesting that fast turnover is crucial
 for its neuroprotective function. We show that STMN2 was primarily
 degraded by the ubiquitin-proteasome system. Its membrane targeting
 N-terminal domain promoted fast turnover, whereas its tubulin binding
 domain promoted stabilization. Proximity labeling and imaging showed that
 tubulin binding reduced STMN2 targeting to trans-Golgi network membranes.
 Pull-down assays showed that tubulin binds preferentially to soluble over
 membrane-bound STMN2. Our observations suggest that STMN2 interconverts
 between a soluble, tubulin-bound form and a membrane-bound, tubulin-free
 form and is rapidly degraded when released from both membranes and
 tubulin. We propose that tubulin binding sequesters and stabilizes STMN2,
 while its neuroprotective function involves an unknown membrane activity.
description: STMN2-V5 expression was induced for 16 hours in U2OS cells. To
 collect the cell lysates from CHX chase assay, the cells were washed with
 cold PBS, harvested, and then lysed using cold EDTA-free NP-40 buffer (50
 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% NP-40 substitute, EDTA-free complete
 protease inhibitor (Roche, 11836170001)), while rocking for 15 minutes at
 4°C. 2 mg of total protein was used for immunoprecipitation. For each
 immunoprecipitation reaction, 70 μL of Dynabeads Protein G (Invitrogen,
 10004D) was coated with 20 μg of rabbit anti-V5 IgG (Novus Biologicals,
 NB600-381), according to the manufacturer’s protocol. Cell lysate and
 coated beads were incubated for 1 hour at 4°C. The Dynabeads were washed
 five times with PBS + 0.1% Triton X-100, and the bound proteins were
 eluted into 40 μL of SDS-PAGE loading buffer. 5 μL of the eluted sample
 was loaded onto each lane for detection by Western blot. 35 μL of the
 eluted sample was loaded onto one lane of SDS-PAGE gel, and submitted to
 Blazin-blue staining (Goldbio P-810-1). Two bands at ~23 KDa and 21 KDa
 were cut and submitted for proteomics analysis. Peptides information was
 uploaded. 250522_All_Peptides.xlsx is a proteomics analysis of the two
 bands of STMN2 expressed in U2OS cells.
description: # Tubulin regulates stability and localization of STMN2 by binding
 preferentially to its soluble form Dataset DOI:
 [10.5061/dryad.j9kd51crx](10.5061/dryad.j9kd51crx) ## Description of the
 data and file structure ### Files and variables #### File:
 250522_All_Peptides.xlsx **Description:** Sheet1, the peptides of STMN2
 from the two bands. Sheet 2 and 3, the peptides of all the proteins
 detected. #### Variables **ScanF #1 / ScanF #2** The scan numbers (or
 spectrum IDs) of the MS/MS spectra from which each peptide was identified.
 These correspond to the individual MS/MS scans in the raw data file. **z
 #1 / z #2** The charge state of the peptide ion (e.g., 2 = doubly
 charged). Higher charge states typically indicate longer or more basic
 peptides. **XCorr #1 / XCorr #2** The cross-correlation score (XCorr) — a
 SEQUEST scoring metric showing how well the observed MS/MS spectrum
 matches the theoretical fragmentation pattern of the peptide. Higher XCorr
 = higher confidence. **ΔCorr #1 / ΔCorr #2** The delta correlation (ΔCn) —
 a measure of how much better the top peptide match is compared to the next
 best match. Values close to 1 mean very strong unique matches; lower
 values mean ambiguity. **Ions #1 / # Ions #2** The number of fragment ions
 matched (observed/theoretical). For example, 7/24 means 7 out of 24
 possible theoretical fragment ions matched. This reflects spectral
 coverage quality. **Reference #1 / Reference #2** The protein accession
 number(s) from a database (e.g., UniProt). Here sp|Q93045|STMN2_HUMAN
 means Swiss-Prot entry Q93045, the human protein STMN2 (Stathmin-2).
 **Redun #1 / Redun #2** The redundancy — how many times this peptide was
 observed or identified in the dataset. Often used to check reproducibility
 or abundance. **Peptide #1 / Peptide #2** The peptide sequences were
 identified. The letters represent amino acids; periods show flanking
 residues from the protein (e.g., R.NINIYTYDDM*EVK.Q means the peptide is
 “NINIYTYDDM*EVK” and the * indicates a modification — likely
 phosphorylation or oxidation). **Gene Symbol #1 / Gene Symbol #2** The
 gene symbols corresponding to the proteins identified — e.g., STMN2. This
 simplifies linking peptides to genes.