Lysosome Associated Protein Transmembrane 4B-24 Is the Predominant Protein Isoform in Human Tissues and Undergoes Rapid, Nutrient-Regulated Turnover

Studies of lysosome associated protein transmembrane 4B (LAPTM4B) have mainly focused on the 35-kDa isoform and its association with poor prognosis in cancers. Here, by employing a novel monoclonal antibody, the authors found that the 24-kDa LAPTM4B isoform predominated in most, both healthy and malignant, human cells and tissues studied. LAPTM4B-24 lacks the extreme N-terminus and, contrary to LAPTM4B-35, failed to promote cell migration. The endogenous LAPTM4B-24 protein was subject to rapid turnover with a t(1/2) of approximately 1 hour. The protein was degraded by both lysosomal and proteasomal pathways, and its levels were increased by the availability of nutrients and lysosomal ceramide. These findings underscore the pathophysiological relevance of the LAPTM4B-24 isoform and identify it as a dynamically regulated effector in lysosomal nutrient signaling.Peer reviewe

A novel estrogen-regulated avian apolipoprotein.

In search for yet uncharacterized proteins involved in lipid metabolism of the chicken, we have isolated a hitherto unknown protein from the serum lipoprotein fraction with a buoyant density of ≤1.063 g/ml. Data obtained by protein microsequencing and molecular cloning of cDNA defined a 537 bp cDNA encoding a precursor molecule of 178 residues. As determined by SDS-PAGE, the major circulating form of the protein, which we designate apolipoprotein-VLDL-IV (Apo-IV), has an apparent Mr of approximately 17 kDa. Northern Blot analysis of different tissues of laying hens revealed Apo-IV expression mainly in the liver and small intestine, compatible with an involvement of the protein in lipoprotein metabolism. To further investigate the biology of Apo-IV, we raised an antibody against a GST-Apo-IV fusion protein, which allowed the detection of the 17-kDa protein in rooster plasma, whereas in laying hens it was detectable only in the isolated ≤1.063 g/ml density lipoprotein fraction. Interestingly, estrogen treatment of roosters caused a reduction of Apo-IV in the liver and in the circulation to levels similar to those in mature hens. Furthermore, the antibody crossreacted with a 17-kDa protein in quail plasma, indicating conservation of Apo-IV in avian species. In search for mammalian counterparts of Apo-IV, alignment of the sequence of the novel chicken protein with those of different mammalian apolipoproteins revealed stretches with limited similarity to regions of ApoC-IV and possibly with ApoE from various mammalian species. These data suggest that Apo-IV is a newly identified avian apolipoprotein

LAPTM4B controls the sphingolipid and ether lipid signature of small extracellular vesicles

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.Peer reviewe

A Ceramide-Regulated Element in the Late Endosomal Protein LAPTM4B Controls Amino Acid Transporter Interaction

Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein's third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.Peer reviewe