Abstract
LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.
Highlights
Leucine zipper transcription factor-like 1 (LZTFL1) is a cytoplasmic and ciliary protein that interacts with other cytosolic proteins, regulates cilia trafficking, and controls β-catenin nuclear localization [1,2,3,4]
The β1 subunit of adaptor protein complex-1 (AP-1) and the clathrin heavy chain were among the proteins identified as LZTFL1-interacting proteins (S1 Table)
Conserved motif DxxFxx[L/F]xxxR was found in proteins that bind to the appendage region of the β2 subunit of adaptor protein complexes (APs)-2 [16, 28], while the dileucine-based motif was shown as the binding site for γ and σ1 of AP-1, α and σ2 of AP-2, and δ and σ3 of AP-3 [28]
Summary
Leucine zipper transcription factor-like 1 (LZTFL1) is a cytoplasmic and ciliary protein that interacts with other cytosolic proteins, regulates cilia trafficking, and controls β-catenin nuclear localization [1,2,3,4]. LZTFL1 is induced by all-trans retinoic acid in activated T cells and associates with the immune synapse [5]. LZTFL1, recently designated as a Bardet-Biedl syndrome protein (BBS), BBS17, binds to BBS9, a constituent of the BBSome complex, and regulates ciliary localization of the BBSome [1]. Knockout of LZTFL1 altered the localization of many proteins of the photoreceptor outer segment [8]. We have shown that adaptor protein complex-1 (AP-1) was abnormally distributed in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 has a role in AP-1-mediated protein trafficking [7]
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