The molecular machinery for lysosome biogenesis

The lysosome serves as a site for delivery of materials targeted for removal from the eukaryotic cell. The mechanisms underlying the biogenesis of this organelle are currently the subject of renewed interest due to advances in our understanding of the protein sorting machinery. Genetic model systems such as yeast and Drosophila have been instrumental in identifying both protein and lipid components of this machinery. Importantly, many of these components, as well as the processes in which they are involved, are proving conserved in mammals. Other recently identified components, however, appear to be unique to higher eukaryotes. BioEssays 23:333-343, 2001. Published 2001 John Wiley & Sons, Inc.     

Maturation models for endosome and lysosome biogenesis

The complexity and dynamic nature of the endocytic apparatus of mammalian cells have become increasingly clear over the past ten years. Structures collectively referred to as endosomes are at the crossroads of traffic with the plasma membrane and with the degradative pathway leading to lysosomes. They carry out the sorting and segregation of receptors and ligands, processes that are necessary for nutrient uptake and the maintenance of plasma membrane composition. This article addresses the question of whether endosomes are stable or transient compartments.     

Role of LAMP-2 in lysosome biogenesis and autophagy

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.     

Higher order Rab programming in phagolysosome biogenesis

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.     

TFEB,a novel mTORC1 effector implicated in lysosome biogenesis,endocytosis and autophagy

Comment on: Peña-Llopis S, et al. EMBO J 2011; 30:3242-58.     

TIP47 is a key effector for Rab9 localization

The human genome encodes approximately 70 Rab GTPases that localize to the surfaces of distinct membrane compartments. To investigate the mechanism of Rab localization, chimeras containing heterologous Rab hypervariable domains were generated, and their ability to bind seven Rab effectors was quantified. Two chimeras could bind effectors for two distinctly localized Rabs; a Rab5/9 hybrid bound both Rab5 and Rab9 effectors, and a Rab1/9 hybrid bound to certain Rab1 and Rab9 effectors. These unusual chimeras permitted a test of the importance of effector binding for Rab localization. In both cases, changing the cellular concentration of a key Rab9 effector, which is called tail-interacting protein of 47 kD, moved a fraction of the proteins from their parental Rab localization to that of Rab9. Thus, relative concentrations of certain competing effectors could determine a chimera's localization. These data confirm the importance of effector interactions for Rab9 localization, and support a model in which effector proteins rely on Rabs as much as Rabs rely on effectors to achieve their correct steady state localizations.     

Energy biogenesis: one key for coordinating two genomes

In metazoan organisms, energy production is the only example of a process that is under dual genetic control: nuclear and mitochondrial. We used a genomic approach to examine how energy genes of both the nuclear and mitochondrial genomes are coordinated, and discovered a novel genetic regulatory circuit in Drosophila melanogaster that is surprisingly simple and parsimonious. This circuit is based on a single DNA regulatory element and can explain both intra- and inter-genomic coordinated expression of genes involved in energy production, including the full complement of mitochondrial and nuclear oxidative phosphorylation genes, and the genes involved in the Krebs cycle.     

Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy

The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to contribute to about 50% of all proteins of the lysosome membrane. Surprisingly, mice deficient in either LAMP-1 or LAMP-2 are viable and fertile. However, mice deficient in both LAMP-1 and LAMP-2 have an embryonic lethal phenotype. These results show that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seems to have more specific functions since LAMP-2 single deficiency has more severe consequences than LAMP-1 single deficiency. Mutations in LAMP-2 gene cause a lysosomal glycogen storage disease, Danon disease, in humans. LAMP-2 deficient mice replicate the symptoms found in Danon patients including accumulation of autophagic vacuoles in heart and skeletal muscle. In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles and unesterified cholesterol, while protein degradation rates are not affected. These results clearly show that the LAMP proteins fulfil functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.     

Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.     

Rab proteins: The key regulators of intracellular vesicle transport

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.     

The Brix domain protein family -- a key to the ribosomal biogenesis pathway?

Six (one archaean and five eukaryotic) protein families have similar domain architecture that includes a central globular Brix domain, and optional N- and obligatory C-terminal segments, both with charged low-complexity regions. Biological data for some proteins in this superfamily suggest a role in ribosome biogenesis and rRNA binding.     

Phylogeny and evolution of Rab7 and Rab9 proteins

Background  

An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.