Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

ESCRT-dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase

YUC flavin monooxygenases catalyze the ratelimiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

Vacuolar system distribution in Arabidopsis tissues,visualized using GFP fusion proteins

Green fluorescent protein (GFP) allows the direct visualization of gene expression and the subcellular localization of fusion proteins in living cells. The localization of different GFP fusion proteins in the secretory system was studied in stably transformed Arabidopsis plants cv. Wassilewskaja. Secreted GFP (SGFP) and GFP retained in the ER (GFP-KDEL) confirmed patterns already known, but two vacuolar GFPs (GFP-Chi and Aleu-GFP) labelled the Arabidopsis vacuolar system for the first time, the organization of which appears to depend on cell differentiation. GFP stability in the vacuoles may depend on pH or degradation, but these vacuolar markers can, nevertheless, be used as a tool for physiological studies making these plants suitable for mutagenesis and gene-tagging experiments.     

Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation

Soluble endoplasmic reticulum (ER)-resident proteins have very long lives because of their ER residency. This residency depends largely on ER-retrieval signals at their C-terminus. We examined the long-term destiny of endogenous ER-resident proteins, a lumenal binding protein (BiP) and a protein disulfide isomerase (PDI), with cultured cells of Arabidopsis. ER residents, in contrast to vacuolar proteinases, were considerably degraded in cells at the stationary phase. A subcellular fractionation analysis suggested that ER residents were transported into the vacuoles, which accumulated the residents lacking the ER-retrieval signals. We showed that the PDI located in the vacuoles had high mannose glycans, but not complex glycans, which suggested that the ER resident was transported to the vacuoles independent of the medial/trans-Golgi complex. To visualize the pathway of transport of ER-resident proteins, tobacco BY-2 cells were transformed with a chimeric gene encoding an ER-targeted green fluorescent protein (30 kDa GFP-HDEL). In the transformed cells at the stationary phase, GFP fluorescence was observed in the vacuoles. A subcellular fractionation revealed that a trimmed form of 27 kDa GFP was localized in the vacuoles. Treatment with E-64d, an inhibitor of papain-type cysteine proteinases that inhibits the degradation of GFP in the vacuoles, resulted in a stable accumulation of 27 kDa GFP in the vacuoles, even in the logarithmic phase. Our results suggest that endogenous ER residents are transported constitutively to the vacuoles by bypassing the Golgi complex and are then degraded.     

Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells

In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through out all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of the SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. Some SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which was defined as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We propose possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.     

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.     

Autophagy-related vacuoles in mouse gallbladder epithelium

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.     

The AtC-VPS protein complex is localized to the tonoplast and the prevacuolar compartment in arabidopsis

Plant cells contain several types of vacuoles with specialized functions. Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown. We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation. VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast. On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants. In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo. These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast. VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments. Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy. We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo. VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.     

Endosomal functions in plants

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins.     

Vacuolar degradation of two integral plasma membrane proteins, AtLRR84A and OsSCAMP1, is cargo ubiquitination-independent and prevacuolar compartment-mediated in plant cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination-independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine-rich repeat receptor-like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans-Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY-2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination-independent manner. Such MVB/PVC-mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination-independent and PVC-mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.     

Green fluorescent protein reveals variability in vacuoles of three plant species

Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.     

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.     

Characterization of Aspergillus nidulans DidBDid2, a non-essential component of the multivesicular body pathway

ESCRT-III heteropolymers mediate membrane protein cargo sorting into multivesicular endosomes for subsequent vacuolar degradation. We studied the localization of largely uncharacterized Aspergillus nidulans ESCRT-III using its key structural component Vps32 and the ‘associated’ component DidB^Did2. Vps32-GFP localizes to motile early endosomes as reported, but predominates in aggregates often associated with vacuoles due to inability to dissociate from endosomes. DidB^Did2 regulating Vps4 (the ATPase disassembling ESCRT-III) is not essential. Consistent with this accessory role, didBΔ is unable to block the MVB sorting of the glutamate transporter AgtA, but increases its steady-state level and mislocalizes a fraction of the permease to the plasma membrane under conditions promoting its vacuolar targeting. didBΔ exacerbates the dominant-negative growth defect resulting from Vps32-GFP over-expression. A proportion of DidB-GFP is detectable in early endosomes colocalizing with RabA^Rab5 and accumulating in nudA1 tips, suggesting that ESCRT-III assembles on endosomes from the early steps of the endocytic pathway.     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

The role of vacuole in plant cell death

Almost all plant cells have large vacuoles that contain both hydrolytic enzymes and a variety of defense proteins. Plants use vacuoles and vacuolar contents for programmed cell death (PCD) in two different ways: for a destructive way and for a non-destructive way. Destruction is caused by vacuolar membrane collapse, followed by the release of vacuolar hydrolytic enzymes into the cytosol, resulting in rapid and direct cell death. The destructive way is effective in the digestion of viruses proliferating in the cytosol, in susceptible cell death induced by fungal toxins, and in developmental cell death to generate integuments (seed coats) and tracheary elements. On the other hand, the non-destructive way involves fusion of the vacuolar and the plasma membrane, which allows vacuolar defense proteins to be discharged into the extracellular space where the bacteria proliferate. Membrane fusion, which is normally suppressed, was triggered in a proteasome-dependent manner. Intriguingly, both ways use enzymes with caspase-like activity; the membrane-fusion system uses proteasome subunit PBA1 with caspase-3-like activity, and the vacuolar-collapse system uses vacuolar processing enzyme (VPE) with caspase-1-like activity. This review summarizes two different ways of vacuole-mediated PCD and discusses how plants use them to attack pathogens that invade unexpectedly.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.     

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.     

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.