RESUMO
Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Glutamina/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Glutamina/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/patologia , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Microambiente TumoralRESUMO
Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.
Assuntos
Hepatócitos/metabolismo , Membranas Intracelulares/química , Fígado/metabolismo , Lisossomos/química , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteoma/isolamento & purificação , Animais , Biomarcadores/metabolismo , Expressão Gênica , Células HeLa , Hepatócitos/química , Humanos , Fígado/química , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Ratos , Ratos WistarRESUMO
Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis.