The kinetochore surveillance phosphatase PP2A-B56 dephosphorylates Knl1 to silence the spindle assembly checkpoint after all chromosomes have been correctly attached to microtubules.

Edited by Phyllis I. HansonThe human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6 -59-aminoacid-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 membranetrafficking 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.ATP-binding cassette (ABC) 2 transporters belong to one of the largest protein families in all organisms (1). In eukaryotes,

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.

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.