differential palmitoylation regulates intracellular patterning of snap25 jennifer greaves and luke...
TRANSCRIPT
Differential palmitoylation regulates intracellular patterning of SNAP25
Jennifer Greaves and Luke H. Chamberlain*
Intro• SNARE proteins regulate exocytosis• Syntaxin 1 and SNAP 25 well studied membrane
proteins that interact w/ vesicle protein VAMP2• SNAP25 pools in recycling endosome (RE) and
trans Golgi network (TGN) shuttled to and from cellular membrane
• Intracellular targeting thought to be regulated by palmitoylation of four cysteine sites on SNAP25
Palmitoylation
• Covalent attachment of palmitate to cysteine residue of protein
• Reversible + post-translational modification• Mediated by palmitoyl transferases DHHC• Hydrophobic anchor…BUT also– Reg. intracellular sorting of protein– Assoc. w/ membrane microdomains– Modulate protein stability
Comparing fluorescent labeled SNAP25 with endogenous SNAP25 and localization in RE and TGN
*PC12 cells
Colocalization of eGFP-SNAP25b and WT SNAP25
• Stained Golgi (GM130), TGN (TGN38), RE (Rab11)• eGFP-SNAP25b
• Quantitative Colocalization Analysis to determine overlap
Colocalization of eGFP-SN25b and RE, TGN, or Golgi
Colocalization of eGFP-SN25b and RE, TGN, or Golgi
• Triple Labelling• Again highest association w/ RE
eGFP-SN25 WT vs. eGFP (85-120)
• Suggests palmitoylation domain is responsible
for RE and TGN targeting and not
interaction w/ any other SNARE proteins.
• Addressed idea that palmitoylation only responsible for membrane anchoring• Added 4CL-KrasMTD to block all cysteines so no binding of palmitate
Palmitoylation mediates targeting of SNAP25 to RE and TGN membranes
• Inhibited protein synthesis w/ Cycloheximide (CHX) to show fluorescence in RE and TGF were from dynamic palmitoylation actions of mature proteins, not newly synthesized ones.
Mutation of single specific palmitoylation sites
Decrease in palmitate incorporation of each cysteine suggests they areAll involved in palmitoylation (C88 being the least affected).
Mutation of single specific palmitoylation site on localization in RE and TGN
• All single cysteine mutations increased targeting to RE and TGN regionswith C88L and C90L showing most dominant effects in directing SNAP25 localization• Localization to RE + TGN is increased when Cys residues decresed from 4 to 3
Cys mutant C88 did not reflect new association with Golgi
• No statistical analysis, difficult to tell any association with just fluorescence.• But, concluded that that no colocalization of eGFP-SN25(C88L) and Golgi
Enhanced intracellular localization of C88 mutant affecting transportation of new
proteins?
Again used CHX to inhibit protein synthesis
Test if C88 mutant was decreasing rate of transportation for new proteins, causing buildup of signal in intracellular targets
NOT buildup, but cycling of palmitoylation affected by mutation
Conclusions
• Palmitoylation can dictate patterning of protein movement across intracellular domains.
• Reversibility of palmitoylation shows how changing # of palmitoylation sites (Cys) regulates localization.
• Cys-rich domain is “autonomous and sufficient” for intracellular localization