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24Mouse Apolipoprotein L9 is a 34-kDa phosphatidylethanolamine (PE)-binding protein. The gene is 25 present only in mouse and rat genomes; hence it is taxonomically restricted. To understand why, it is 26 essential to uncover details about its functions in cellular processes. Here we show that ApoL9 27 interacts with the proteins of the LC3 and GABARAP subfamilies, which are key players in 28 macroautophagy. In amino-acid starved cells it preferentially interacts with lipidated LC3B, likely by 29 binding to its PE moiety. On treatment with autophagy inhibitors bafilomycin A1 and chloroquine, 30ApoL9 is found near swollen mitochondria and on lysosomes/LAMP1-positive compartments. 31However, ApoL9 itself does not seem to be degraded as a result of autophagy, suggesting that it is 32 not an autophagy cargo receptor. Deletions in a putative transmembrane region between amino 33 acids 110 and 145 abolish PE-binding. In addition, ApoL9 can redistribute to stress granules, can 34 homooligomerize, and is a microtubule-associated protein. In short, its distribution in the cell is 35 quite widespread, suggesting that it could have functions at the intersection of membrane binding 36 and reorganization, autophagy, cellular stress and intracellular lipid transport. 37 38 Summary statement 39 This article is about how Apolipoprotein L9, a lipid-binding protein, has versatile properties and 40 influences a variety of processes taking place inside an animal cell. 41 42 43 44 45 46 47 48 50 Mouse ApoL9, encoded by two independent genes Apol9a and Apol9b on chromosome 15, has 51previously been shown to have either antiviral or pro-viral effects during infection of cells by 52 different types of viruses (Kreit et al., 2014;Kreit et al., 2015;Arvind and Rangarajan, 2016). 53Expression of ApoL proteins is also induced by interferons and TNF-Zhaorigetu et al., 2011, 54 Monajemi et al., 2002. Small quantities of ApoL9 secreted from macrophages during interferon 55 induction have been shown to promote epithelial cell proliferation in a paracrine fashion (Sun et al., 56 2015). However, most of the protein is retained within the cell. In a previous study, we used B16F10 57 melanoma cells to look at the basic expression pattern of constructs expressing ApoL9, examined its 58 levels in various mouse tissues, and viewed it in the context of infection by Japanese Encephalitis 59 virus (Arvind and Rangarajan, 2016). We reported that ApoL9 is a phosphatidylethanolamine-binding 60 protein that, in normal conditions, has a general cytoplasmic distribution and can localize to 61 ubiquitin-positive bodies called ALIS (aggresome-like induced structures) and aggresomes. ApoL9 is 62 expressed at moderate to high levels in mouse liver and brain, suggesting some function of 63 relevance for the protein in these major tissues. 64In order to understand the functions of ApoL9, it is essential to know more about its distribution in 65 the cell and the proteins it interacts with. In this study, our objective is to attempt to uncover as 66 many clue...

Mouse Apolipoprotein L9 is a 34-kDa phosphatidylethanolamine (PE)-binding protein. The gene is present only in mouse and rat genomes; hence it is restricted to two species. To understand why, it is essential to uncover details about its functions in cellular processes. Here we show that ApoL9 interacts with the proteins of the LC3 and GABARAP subfamilies, which are key players in macroautophagy. In vitro binding studies show a strong association with GABARAP, and in amino acid-starved cells it preferentially interacts with lipidated LC3B, likely by binding to its PE moiety through its lipid-binding domain. On treatment with autophagy inhibitors bafilomycin A1 and chloroquine, ApoL9 is found near swollen mitochondria and on lysosomes/LAMP1-positive compartments. However, ApoL9 itself does not seem to be degraded as a result of autophagy, suggesting that it is not an autophagy cargo receptor. Deletions in a putative transmembrane region between amino acids 110 and 145 abolish binding to PE. In addition, ApoL9 can redistribute to stress granules, can homo-oligomerize, and is a microtubule-associated protein. In short, its distribution in the cell is quite widespread, suggesting that it could have functions at the intersection of membrane binding and reorganization, autophagy, cellular stress and intracellular lipid transport.

Map1Lc3b is a protein that has pivotal functions in cellular autophagy. At least three groups in the past decade have reported its presence in the nucleoli of cells, but its functions in that organelle remain unknown. We isolated a few clonal populations of cells stably expressing V5-tagged mouse Lc3b highly enriched in the nucleoli, but the frequency of occurrence of such clones was strikingly low. The phenomenon was readily reproducible, though the protein in the nucleolus puzzlingly had varying molecular masses in different clones but consistently displayed a very strong interaction with the mitochondrial protein C1qbp, which has well-documented functions in the nucleolus. We investigated further and discovered that, in at least one of the clones, Lc3b had formed a chimera with the puromycin resistance gene in the plasmid, plausibly by illegitimate recombination during or after integration of the construct into the cellular genomic DNA. The -1 shifted reading frame of puromycin N-acetyltransferase (pac) can encode a protein that is equally long as the one encoded by the complete pac ORF, but is targeted to the nucleoli due to a drastic shift in the isoelectric point (pI). Notably, this set of events again brings into focus the low threshold often reported for recombination events to occur in eukaryotic cells, the multiple factors influencing them, and calls for increased vigilance in experiments involving DNA transfection and gene targeting.