Tubulin regulates stability and localization of STMN2 by binding preferentially to its soluble form

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 ac..., , 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 Bl..., # 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 ...