5-phosphatase domain of Lowe oculocerebrorenal
syndrome protein (OCRL) fused to cryptochrome
2 (CRY2) (27, 28) did not affect the localization
of TMEM24 to contact sites in COS-7 cells (Fig. 3A).
Thus, TMEM24 binds the PM selectively because
the cytoplasmic leaflet of this membrane is highly acidic owing to an enrichment of PS and phosphoinositides, which function redundantly.
Consistent with the data obtained in living cells,
we found that lipid bilayer binding in vitro was
dependent on the C-terminal portion of TMEM24
because a fragment comprising only the SMP
and C2 domains (residues 76 to 414) did not bind
acidic liposomes. Nor did the same construct bind
liposomes in the presence of calcium (Fig. 2H),
which induces membrane interactions for a number of previously characterized C2 domains (29).
Sequences in the TMEM24 C terminus are highly
conserved (fig. S2B), indicative of functional importance, and are enriched in basic residues, which
are well suited to mediate interactions with acidic
membranes.
Regulation of TMEM24 localization by
calcium-dependent phosphorylation
and dephosphorylation
The critical role of calcium dynamics in regulated
secretion and the previously observed requirement of TMEM24 for efficient insulin release (13)
prompted us to investigate whether calcium regulates TMEM24 localization. To this end, we treated
HeLa cells transfected with TMEM24-EGFP with
thapsigargin, which elevates cytosolic calcium
levels by blocking the ER calcium pump (SERCA)
Lees et al., Science 355, eaah6171 (2017) 17 February 2017 5 of 14
Fig. 4. TMEM24 harbors a phosphatidylinositol (PI)–binding SMP mod-
ule. (A) TMEM24 (76 to 260) adopts an SMP domain fold. TMEM24 SMP
domain is modeled as a dimer by analogy to the E-Syt2 SMP domains (at
right). Disordered segments are represented as dashed lines. TMEM24 helix
a2 and strand b13 move closer compared with the corresponding helix and
strand in the liganded E-Syt2 structure. Four lipid molecules bound by E-Syt2
dimer are indicated. (B) Native electrospray ionization mass spectrometry
(ESI-MS) analysis of TMEM24 SMP domain purified from Expi293 cells indi-
cates dimerization, with masses consistent with one phospholipid molecule
bound per monomer. Whereas the monomeric fraction exists in both apo and
holo form, the dimeric fraction is mostly complexed with lipid. (C) SEC of
purified TMEM24 SMP domain preincubated with liver PI indicates that PI
binds and comigrates with TMEM24 SMP. Although TMEM24 SMP migrates
as a dimer in solution, a small shift in apparent molecular weight upon binding
to PI occurs, consistent with a change in conformation but not oligomerization
state. (D) Analysis of individual fractions from 14.0 to 16.0 ml by SDS-PAGE
confirms redistribution of the liganded SMP domain. (E) Fractions between
14.0 and 16.0 ml retention volume were pooled; lipid was extracted and
resolved by thin-layer chromatography. The PI band did not appear in these
fractions when TMEM24 SMP or liver PI was subjected separately to SEC.
