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

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 neu..., 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..., , # 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\
Norm...,