ABCMdb

PMID: 20863830

Engelbrecht S, Kaltenborn E, Griese M, Kern S
The surfactant lipid transporter ABCA3 is N-terminally cleaved inside LAMP3-positive vesicles.
FEBS Lett. 2010 Oct 22;584(20):4306-12. Epub 2010 Sep 21., [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Two ABCA3 point mutations Q215K and E292V were introduced into WT hABCA3 by site-directed mutagenesis (Stratagene). Login to comment 90 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys We used two ILD-related ABCA3 mutations (Supplementary Fig. 1): Q215K, a misfolding defect studied here for the first time, and E292V, a functional mutation [10], to prove that the ER retention in general, regardless of the cause (mutation as intrinsic cause or enforced processing impairment, Fig. 2) hinders the ABCA3 maturation. Login to comment 91 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Localization studies in A549 cells stably expressing WT, Q215K or E292V proteins with C-terminal HA-tag showed that the E292V mutant colocalized properly with the MVB/LB protein LAMP3 (Fig. 4A) but not with the ER protein calnexin (Fig. 4B). Login to comment 92 ABCA3 p.Gln215Lys Q215K mutant remained in the ER compartment, overlapping with calnexin (Fig. 4B) and showing no colocalization with LAMP3 (Fig. 4A). Login to comment 93 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys Immunodetection of WT, Q215K and E292V proteins via HA-tag demonstrated that the ER retained Q215K protein completely lacked the 150 kDa band, which was strongly present in the MVB/LB-localized WT Fig. 2. Login to comment 98 ABCA3 p.Glu292Val Results of three independent experiments are presented; *P < 0.05, **P < 0.01, ***P < 0.001. and E292V proteins (Fig. 4C). Login to comment 121 ABCA3 p.Gln215Lys Moreover, strong ABCA3 ER retention caused by Q215K mutation resulted in vanishing of the lower band (Fig. 4), showing that the effect was not specific only for the chemical interference of the ABCA3 processing. Login to comment 124 ABCA3 p.Gln215Lys The ER-retained Q215K mutant lacks the 150 kDa band. Login to comment 125 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys WT ABCA3 and two mutants E292V and Q215K (all C-terminal HA-tag) were stably expressed in A549 cells. Login to comment 126 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys HA-tag immunofluorescence of WT and E292V (green) showed their correct LAMP3-colocalization (A, red) while Q215K mutant (green) remained in the ER colocalizing with calnexin (B, red). Login to comment 127 ABCA3 p.Gln215Lys (C) ABCA3 immunodetection in cell lysates via anti-HA-tag antibody showed absence of the 150 kDa band in the case of the Q215K protein. Login to comment 134 ABCA3 p.Leu101Pro ABCA3 p.Gln215Lys complete absence of the lower band we noticed only in the case of Q215K and other ER retained ABCA3 mutations (e.g. L101P; own data, [8,9]). Login to comment 137 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys The ER retained mutation Q215K, which completely lacks the 150 kDa band, does not support the biogenesis of LAMP3-positive vesicles (Fig. 4, red signal) in A549 cells, while WT protein and even E292V mutant that has reduced ATP hydrolysis capacity [10] both do. Login to comment 138 ABCA3 p.Gln215Lys Thus it appears that the presence of only the 190 kDa band (as for Q215K mutant) within A549 cells is not enough for ABCA3 function, and thus ABCA3 maturation is indispensible for its role. Login to comment 139 ABCA3 p.Glu292Val Instead accumulation of the lower 150 kDa band, a process that includes N-terminal cleavage, is necessary for at least partial ABCA3 function (Fig. 4, E292V mutation). Login to comment