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

Additional Cys incur SNAP23 activity?

Fluorescence ratio = 1.0 +/- 0.022 indicating no change.

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

Role of Cys-rich domain directing intracellular localization of SNAP25

Role of Cys-rich domain directing intracellular localization of SNAP25

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

Relevence

• Palmitoylation’s unique dynamics to protein localization.

• Example of good use of controls, covering all the bases.