| Autor: |
Tuomivaara ST; Department of Laboratory Medicine, University of California, San Francisco, CA., Teo CF; Howard Hughes Medical Institute, University of California, San Francisco, CA., Jan YN; Howard Hughes Medical Institute, University of California, San Francisco, CA.; Department of Physiology, University of California, San Francisco, CA.; Department of Biochemistry and Biophysics, University of California, San Francisco, CA., Jan LY; Howard Hughes Medical Institute, University of California, San Francisco, CA.; Department of Physiology, University of California, San Francisco, CA.; Department of Biochemistry and Biophysics, University of California, San Francisco, CA., Wiita AP; Department of Laboratory Medicine, University of California, San Francisco, CA.; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA.; Chan Zuckerberg Biohub, San Francisco, CA. |
| Abstrakt: |
To facilitate our understanding of the often rapid and nuanced dynamics of extracellularly exposed proteomes during signaling events, it is important to devise robust workflows affording fast time resolution without biases and confounding factors. Here, we present S urface-exposed protein La beling using P eroxida S e, H 2 O 2 , and T yramide-derivative (SLAPSHOT), to label extracellularly exposed proteins in a rapid, sensitive, and specific manner, while preserving cellular integrity. This experimentally simple and flexible method utilizes recombinant soluble APEX2 peroxidase that is applied to cells, thus circumventing biological perturbations, tedious engineering of tools and cells, and labeling biases. APEX2 neither requires metal cations for activity nor contains disulfide bonds, conferring versatility for a wide spectrum of experimental setups. We applied SLAPSHOT followed by quantitative mass spectrometry-based proteomics analysis to examine the immediate and extensive cell surface expansion and ensuing restorative membrane shedding upon the activation of Scott syndrome-linked TMEM16F, a ubiquitously expressed calcium-dependent phospholipid scramblase and ion channel. Time-course data ranging from one to thirty minutes of calcium stimulation using wild-type and TMEM16F deficient cells revealed intricate co-regulation of known protein families, including those in the integrin and ICAM families. Crucially, we identified proteins that are known to reside in intracellular organelles, including ER, as occupants of the freshly deposited membrane, and mitovesicles as an abundant component and contributor to the extracellularly exposed proteome. Our study not only provides the first accounts of the immediate consequences of calcium signaling on the extracellularly exposed proteome, but also presents a blueprint for the application of SLAPSHOT as a general approach for monitoring extracellularly exposed protein dynamics. |
