Lysosomal targeting of the ABC transporter TAPL is determined by membrane-localized charged residues.

The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6-59-amino-acid-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain, TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear. Here, we delineated the route of TAPL to lysosomes and investigated the determinants of single trafficking steps. By synchronizing trafficking events by a retention using selective hooks (RUSH) assay and visualizing individual intermediate steps through immunostaining and confocal microscopy, we demonstrate that TAPL takes the direct route to lysosomes. We further identified conserved charged residues within TMD0 transmembrane helices that are essential for individual steps of lysosomal targeting. Substitutions of these residues retained TAPL in the endoplasmic reticulum (ER) or Golgi. We also observed that for release from the ER, a salt bridge between Asp-17 and Arg-57 is essential. An interactome analysis revealed that Yip1-interacting factor homolog B membrane-trafficking protein (YIF1B) interacts with TAPL. We also found that YIF1B is involved in ER-to-Golgi trafficking and interacts with TMD0 of TAPL via its transmembrane domain and that this interaction strongly depends on the newly identified salt bridge within TMD0. These results expand our knowledge about lysosomal trafficking of TAPL and the general function of extra transmembrane domains of ABC transporters.

Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.

Team work at its best ­ TAPL and its two domains.

The transporter associated with antigen processing (TAPL, ABCB9) is a homodimeric ABC transporter, shuttling cytosolic polypeptides into the lumen of lysosomes energized by ATP hydrolysis. Here we give a short overview of the superfamily of ABC transporters and summarize the current state of knowledge on TAPL in detail. The architecture of TAPL and its substrate specificity are described and we discuss the function of an extra N-terminal transmembrane domain, called TMD0, in respect of subcellular targeting and interaction with proteins, contributing to long-term stability. As TAPL shows ­ besides a ubiquitous basal expression ­ an elevated expression in antigen presenting cells, we present models of TAPL function in adaptive immunity.

PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing.

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.