Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes

Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.     

Membrane dynamics and the biogenesis of lysosomes

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

A melanosome-associated monoclonal antibody J1 recognizes luminal membrane of prelysosomes common to biogenesis of melanosomes and lysosomes

Melanogenesis cascade may be directly or indirectly linked to the dynamics of endosome-lysosome biogenesis. This study aims to identify how and to what extent the endosome-lysosome system is involved in melanosome biogenesis, by utilizing a novel melanogenesis marker, J1, which we identified in the process of developing monoclonal antibodies (MoAbs) against human melanosomes. The antigenic epitope of MoAb J1 was expressed by all of the melanotic and nonmelanotic cells examined. It was expressed primarily by granular structures located in regions proximal to the Golgi complex. Most of MoAb J1 positive granules were co-stained with melanogenic markers, tyrosinase or tyrosinase-related protein (TRP-1). The epitope of MoAb J1 was also coexpressed by most, but not all, of LGP85 (a lysosomal marker) positive granules in both melanoma and non-melanoma cells, indicating that MoAb J1 recognizes a subset of lysosomal vesicles. MoAb J1 did not, however, react with vesicles with late/early (syntaxin 8/ EEA1) endosomal markers. Further examination using fluorophore-labeled pepstatin, a marker of lysosomal luminal content, confirmed that MoAb J1 specifically recognizes the luminal surface of lysosomes. These results indicate that MoAb J1 possesses an antigen epitope that is expressed in the luminal component of prelysosomal granules which are involved in the biogenesis cascade common to both melanosomes and lysosomes. We suggest that tyrosinase family protein, tyrosinase and TRP-1 are transported to melanosomes from TGN via these prelysosomal granules after being transiently transported to late endosomes.     

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.     

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.     

Lysosomal acid phosphatase is transported via endosomes to lysosomes.

Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1 + 2), enriched in endosomes. The Golgi fraction (GF1 + 2) was prepared from the livers of rats given [35S]methionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3'-diaminobenzidine (DAB) and H2O2 reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes.     

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.     

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L ‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy‐dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.     

Thylakoid membrane proteomics

Proteomics seeks to monitor the global complement of proteins within a cell or organism and accompanying plasticity with respect to development and environment. The proteome is dynamic, the product of current and past gene expression, countless protein-protein interactions and selective proteolytic systems. Consequently the snapshot that a proteomic measurement yields must be integrated into proteome flux; the flow of nutrients and energy through the protein pathways that catalyze and drive life. The thylakoid membrane proteome poses many technical challenges for proteomics. Integral membrane proteins present awkward physico-chemical properties and the abundant photosynthetic machinery conceals much less abundant and no less important proteins such as channels and transporters that control the interaction of stroma and lumen. Discussed here are contrasting approaches to thylakoid proteomics; 'shotgun' techniques that provide throughput benefits by cleaving proteins into smaller more-manageable peptide chunks versus intact protein techniques that provide more detailed and accurate pictures. A two-dimensional chromatography system directly interfaced to electrospray-ionization mass spectrometry has allowed the direct visualization of large reaction-center proteins (up to 83 kDa) from both Photosystems 1 and 2 providing an attractive avenue for characterization of thylakoid membrane proteomes under different conditions because of the ability to resolve molecular heterogeneity resulting from post-translational modifications such as phosphorylation and oxidation. A high-resolution spectrum of Bacteriorhodopsin recorded to an accuracy of 8 ppm using Fourier-transform mass spectrometry demonstrates the first application of this technique to intact polytopic integral membrane proteins.     

Bacterial membrane proteomics

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.     

Plant membrane proteomics

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.     

Cracking outer membrane biogenesis

The outer membrane is a distinguishing feature of the Gram-negative envelope. It lies on the external face of the peptidoglycan sacculus and forms a robust permeability barrier that protects extracytoplasmic structures from environmental insults. Overcoming the barrier imposed by the outer membrane presents a significant hurdle towards developing novel antibiotics that are effective against Gram-negative bacteria. As the outer membrane is an essential component of the cell, proteins involved in its biogenesis are themselves promising antibiotic targets. Here, we summarize key findings that have built our understanding of the outer membrane. Foundational studies describing the discovery and composition of the outer membrane as well as the pathways involved in its construction are discussed.     

Membrane proteins and membrane proteomics

Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins.     

Lysosomal acid phosphatase is transported to lysosomes via the cell surface.

Lysosomal acid phosphatase (LAP) is transported as a transmembrane protein to dense lysosomes. The pathway of LAP to lysosomes includes the passage through the plasma membrane. LAP is transported from the trans-Golgi to the cell surface with a half-time of less than 10 min. Cell surface LAP is rapidly internalized. Most of the internalized LAP is transported back to the cell surface. On average, each LAP molecule cycles greater than 15 times between the cell surface and the endosomes before it is transferred to dense lysosomes. At equilibrium approximately 4 times more LAP precursor is present in endosomes than at the cell surface. Exposing cells to reduced temperature or weak bases such as NH4Cl, chloroquine and primaquine decreases the steady-state concentration of LAP at the cell surface. The recycling pathway is operative at greater than or equal to 20 degrees C and does not include passage of the Golgi/trans-Golgi network. LAP is transferred with a half-time of 5-6 h from the plasma membrane/endosome pool to dense lysosomes, from where it does not recycle to the endosome/plasma membrane pool at a measurable rate.     

Lysosomal proteinase activity and the the lysosomes of the small intestine in postnatal ontogeny

It has been shown that in postnatal animals, the activity of lysosomal proteinases in the distal part of the small intestine was considerably higher than in the proximal one. With transition from milk to definitive nutrition, the activity of lysosomal proteinases gets equalized in the proximal and distal parts of the small intestine. The data concerning the proximo-distal gradient of distribution of the activity of lysosomal proteinases have been supported. Transition from milk to definitive nutrition has given rise to ultrastructural alterations in the lysosomal apparatus.