Abstract
BackgroundThe membrane anchored kinase, LMTK2, is a serine/threonine kinase predominantly localized to endosomal compartments. LMTK2 has been shown to be involved in the trafficking of the CFTR ion channel, the androgen receptor, as well as modulating neurodegeneration. As a membrane anchored protein, LMTK2 must be exported from the ER, yet the mechanisms whereby LMTK2 is sequestered within the ER for efficient export are unknown.MethodsSequence analysis of the carboxyl tail of LMTK2 revealed a putative di-acidic ER export motif. Site-directed mutagenesis was utilized to ablate this potential motif. Subcellular fractionation, immunofluorescence microscopy, and transferrin recycling assays were used to determine the consequence of mutating LMTK2’s export motif.ResultsMutation of the di-acidic export motif led to ER retention of LMTK2, and an increase in protein half-life and a concomitant loss of LMTK2 from its appropriate terminal destination. Loss of LMTK2 from endosomal compartments by preventing its release from the ER is linked to a reduction in transferrin recycling.ConclusionsWe have identified a di-acidic ER export motif within the carboxyl tail of the membrane anchored kinase LMTK2. This sequence is used by LMTK2 for its efficient export from the ER.
Highlights
The membrane anchored kinase, Lemur tyrosine kinase 2 (LMTK2), is a serine/threonine kinase predominantly localized to endosomal compartments
LMTK2 contains potential acidic endoplasmic reticulum (ER) export motifs Alignment of the sequences of various mammalian orthologs of LMTK2 revealed the presence of a canonical diacidic motif in the distal portion of the C-terminal tail (Fig. 1a, b), irrespective of the overall length of each species LMTK2
To explore the possible role acidic amino acids in the efficient export of LMTK2 from the ER, we studied the trafficking of wild-type and mutant LMTK2 (A1110SA), where we mutated the D1110SE of sequence of wild-type LMTK2 to A1110SA
Summary
The membrane anchored kinase, LMTK2, is a serine/threonine kinase predominantly localized to endosomal compartments. Initiated by the binding of GTP to the small GTPase Sar, the association of other coat proteins (COPs) Sec and Sec, forms a pre-budding complex that yields a concave surface enriched in basic residues [1,2,3]. Thought to be a bulk flow process [4], the efficient export of proteins from the endoplasmic reticulum (ER) is thought to be a selective process [5], whereby the Sar1-Sec23/24 complex elicits membrane curvature, and selectively enriches cargo proteins into the newly forming COP vesicle. Several groups have identified specific amino acid sequences within the cytoplasmic domains of newly synthesized (and properly folded) proteins that act as called ER export motifs [6,7,8]. Of note for VSV-G, an upstream tyrosine residue appears to enhance the activity of the di-acidic motif (501YTDIE) [10]
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