Whereas we have a profound understanding about the function and biogenesis of the protein constituents in the lumen of the lysosomal compartment, much less is known about the functions of proteins of the lysosomal membrane. Proteomic analyses of the lysosomal membrane suggest that, apart from the well-known lysosomal membrane proteins, additional and less abundant membrane proteins are present. The identification of disease-causing genes and the in-depth analysis of knockout mice leading to mutated or absent membrane proteins of the lysosomal membrane have demonstrated the essential role of these proteins in lysosomal acidification, transport of metabolites resulting from hydrolytic degradation and interaction and fusion with other cellular membrane systems. In addition, trafficking pathways of lysosomal membrane proteins are closely linked to the biogenesis of this compartment. This is exemplified by the recent finding that LIMP-2 (lysosomal integral membrane protein type-2) is responsible for the mannose 6-phosphate receptor-independent delivery of newly synthesized β-glucocerebrosidase to the lysosome. Similar to LIMP-2, which could also be linked to vesicular transport processes in certain polarized cell types, the major constituents of the lysosomal membrane, the glycoproteins LAMP (lysosome-associated membrane protein)-1 and LAMP-2 are essential for regulation of lysosomal motility and participating in control of membrane fusion events between autophagosomes or phagosomes with late endosomes/lysosomes. Our recent investigations into the role of these proteins have not only increased our understanding of the endolysosomal system, but also supported their major role in cell physiology and the development of different diseases.
- cholesterol homoeostasis
- lysosome membrane protein
The lysosomal membrane: a unique intracellular membrane displaying multiple functions
The lysosomal membrane, a 7–10-nm-thick single phospholipid bilayer, separates the potent catabolic enzymes from other cellular constituents, protecting them from degradation. However, the lysosomal membrane represents more than a mechanical border. It has to fulfil a large number of different functions, including the establishment of the pH gradient between the lysosomal lumen and cytoplasm, mediation of fusion with endosomes or other organelles, sequestration of lysosomal enzymes and the transport of degradation products into the cytoplasm [1,2]. It controls the passage of material across the lysosomal membrane by mediating fusion with other organelles as well as selective transport processes. Kinetic efflux and influx studies of radiolabelled metabolites into lysosomes were used to demonstrate the existence of more than 20 lysosomal membrane transporters . The extremely high carbohydrate content is a unique feature of the lysosomal membrane. Within their luminal domain, the integral proteins of this membrane are heavily glycosylated with few O-glycans and many N-glycans, most of which are of the complex poly-N-acetyl-lactosamine type . LAMP (lysosome-associated membrane protein)-1, LAMP-2, LIMP (lysosomal integral membrane protein)-2 and CD63 are the most abundant components of this membrane. In addition to these major proteins, a number of less abundant or transient components of the lysosomal membrane have been described . The biosynthetic route of lysosomal membrane proteins from the rough ER (endoplasmic reticulum) to the lysosomal compartment has been extensively studied in recent years . In particular, tyrosine- or leucine-containing sorting signals, usually residing in the cytoplasmic domains of these proteins, have been identified and characterized and found to be crucial for lysosomal transport. However, there is increasing evidence for the existence of multiple pathways for the delivery of hydrolases and membrane proteins to the lysosome . It is becoming apparent that lysosomal membrane proteins contribute to various functions along their transport route. Although it is known that mutations in predicted membrane proteins are associated with human diseases (e.g. neuronal ceroid lipofuscinosis, Niemann–Pick disease; see Figure 1), the function of most of them still has to be discovered. An increasing number of gene mutations that lead to lysosomal dysfunction and disease have been identified. Studies using knockout mice and non-mammalian model organisms underline further the importance of these proteins.
Functions of LAMPs
The lysosomal membrane proteins LAMP-1 and LAMP-2 are estimated to constitute approx. 50% of all proteins of the lysosome membrane. The viability of mice deficient in either LAMP-1 or LAMP-2, as well as the embryonic lethal phenotype of LAMP-1/LAMP-2 double-deficient mice strongly suggest that LAMPs share common functions in vivo . However, since LAMP-2 single deficiency has more severe consequences, including accumulation of autophagic vacuoles in various tissues, LAMP-2 seems to have more unique functions than LAMP-1. LAMP-2-deficient mice replicate the symptoms found in human patients suffering from Danon disease, a fatal cardiomyopathy and myopathy, including accumulation of autophagic vacuoles in heart and skeletal muscle . The central role of LAMP-2 is also underlined by a recent study where LAMP-2-knockout mice were shown to have an impaired phagosomal maturation in neutrophils . The impairment of this important innate immune defence process in these mice leads to periodontitis. The retarded clearance of bacterial pathogens was found to be due to an inefficient fusion capacity between lysosomes and phagosomes. Interestingly, a specific type of autophagy, where cytosolic proteins are transported directly into the lysosome for degradation, requires an isoform of LAMP-2 (LAMP2A) as a receptor . Substrates of this CMA (chaperone-mediated autrophagy) are a subset of cytosolic proteins with a motif recognized by the hsc70 (heat-shock cognate 70) chaperone. The chaperone–substrate complex then binds to LAMP2A acting as a CMA receptor. After unfolding, the substrate crosses the lysosomal membrane assisted by a luminal chaperone (lys-hsc70) and is rapidly degraded. CMA is maximally activated during stress such as prolonged starvation, mild oxidation and other conditions resulting in protein damage. CMA activity decreases during aging and in some age-related disorders such as familial forms of Parkinson's disease .
In embryonic fibroblasts, mutual disruption of both LAMPs is associated with an increased accumulation of autophagic vacuoles, altered lysosomal appearance and disturbed cholesterol metabolism, whereas protein degradation rates are not affected . The double deficiency in mice leads to embryonic lethality at day 15.5, providing in vivo evidence that LAMP-1 and LAMP-2 have overlapping functions which are at least in part independent of CMA. This is also underlined by recent studies where cells lacking either one or both LAMPs were analysed for phagolysosome biogenesis [13,14]. Whereas macrophages and fibroblasts from single-deficient mice display normal fusion of lysosomes with phagosomes, in LAMP-double-knockout fibroblasts, phagosomes are unable to recruit late endosomal/lysosomal markers, and phagocytosis is arrested before the acquisition of Rab7. Interestingly, the maturation of Neisseria-containing phagosomes is also disturbed, and mouse fibroblasts lacking both LAMPs fail to kill the engulfed pathogen . Also the starvation-induced maturation of autophagosomes is disturbed in double-deficient fibroblasts, LAMP-2-deficient hepatocytes and, to a lesser extent, in LAMP-2-depleted HeLa cells [15,16]. The maturation block caused by LAMP deficiency is at least partially due to the failure of (auto)phagosomes to move towards lysosomes along microtubules via dynein/dynactin-mediated centripetal displacement . In addition, LDL (low-density lipoprotein) receptor and LDL uptake are increased in LAMP-deficient cells, accompanied by a defect in esterification of both endogenously synthesized and LDL-derived cholesterol. These data suggest that LAMP-deficient cells are defective in cholesterol transport to the site of its esterification in the ER, probably due to defective export of cholesterol out of late endosomes or lysosomes . It was found that LAMP-2, and its luminal domain in particular, plays a critical role in endosomal cholesterol transport which is distinct from the CMA function of LAMP-2.
LIMP-2: an intriguing lysosomal membrane protein with multiple functions
LIMP-2/LGP85 belongs to the CD36 family of scavenger receptors and is one of the most abundant ubiquitously expressed lysosomal membrane proteins. It spans the membrane twice, with the N- and C-terminus located in the cytosol and exhibits a highly glycosylated loop within the lysosomal lumen . LIMP-2 may be involved in lysosome/endosome biogenesis . Overexpression of LIMP-2/LGP85 was shown to result in the accumulation of large swollen vacuoles that share both early and late endosomal as well as lysosomal features. These large vacuoles appear electron-lucent with only occasional luminal membranes, suggesting that the invagination of internal vesicles may be impaired. Pulse–chase experiments showed that the large vacuoles were not initially derived from lysosomes. Co-expression of dominant-negative Rab5b with LIMP-2/LGP85 totally inhibited the formation of the large swollen vacuoles, indicating that normal function of Rab5 was necessary for their appearance. These results suggest that LIMP-2/LGP85 may control the balance between vesicle invagination and vesicle budding from the limiting membrane of endosomal compartments. It is possible that overexpression of LIMP-2/LGP85 causes a dispersal of the budding machinery, which might be due to an impaired recruitment of an as-yet-unknown cytoplasmic factor that is involved in vesicular fission and/or fusion. LIMP-2-deficient mice  are characterized by an increased postnatal mortality rate which was associated with the development of an unilateral or bilateral hydronephrosis caused by an obstruction of the ureteropelvic junction. An accumulation of lysosomes in the epithelial cells of the ureter adjacent to the ureteral lumen and a disturbed apical expression of uroplakin was observed, suggesting an impairment of membrane transport processes. Serious hearing defects in LIMP-2-deficient animals were indicated by deficits in acoustic startle responses, in brainstem evoked auditory potentials and a reduced potassium concentration in the endolymph of the inner ear. LIMP-2-deficient mice suffer from a massive decline of spiral ganglia in the cochlea concomitant with a loss of the inner and outer hair cells. These pathological changes begin at the age of 3 months and are probably secondary to a degeneration of the stria vascularis . LIMP-2-deficient mice are also characterized by a peripheral demyelinating neuropathy. Demyelination was associated with a massive loss of peripheral myelin proteins and an increased activity and expression of lysosomal proteins, highlighting a hitherto unknown role of LIMP-2 for myelin maintenance.
It was also shown that LIMP-2 is the receptor for the mannose 6-phosphate receptor-independent transport of β-GC (β-glucocerebrosidase) to the lysosome . In LIMP-2-deficient fibroblasts or macrophages, β-GC is no longer effectively transported to the lysosome, but is secreted into the cell culture medium. Also, in vivo missorting of β-GC occurs with low tissue levels of β-GC and increased enzyme activity in serum of LIMP-2-deficient mice. Our previous studies indicate that the interaction of β-GC and LIMP-2 takes place very early in the secretory pathway in the ER. From the ER, the receptor–ligand complex then traffics through the Golgi to the lysosome, where its acidic pH probably leads to a dissociation of the ligand from its receptor. Recently, mutations in the human gene encoding the lysosomal integral membrane protein LIMP-2 were found to be responsible for AMRF (action myoclonus–renal failure) syndrome, a fatal autosomal-recessive disorder characterized by focal glomerulosclerosis, progressive myoclonus epilepsy and ataxia . It was found that AMRF disease-causing mutations similar to a disruption of a crucial coiled-coil domain within the luminal part of LIMP-2 abolished β-GC binding .
CD63: an unusual lysosomal tetraspanin
CD63, also called LIMP-1, belongs to the family of tetraspanins . This family is composed of 33 members in mammals, spanning the membrane four times and forming a small and a large extracellular loop. Tetraspanins group specific cell-surface proteins and thereby increase the formation and stability of functional signalling complexes. Such complexes are involved in diverse cellular processes, such as cell activation, adhesion, motility, differentiation and malignancy. CD63 is an exceptional tetraspanin, since, at steady state, it is usually found as a heavily glycosylated protein in late endosomes/lysosomes. The majority of tetraspanins described so far usually reside in the plasma membrane. Despite the existence of abundant data on the presumed role of CD63 in isolated cell types, its function in vivo is largely unknown. The phenotype of CD63-knockout mice  suggests a role for CD63 in development and distribution of immune system cells, a regulatory activity in platelet adhesion, and an important role in kidney physiology.
Towards a complete understanding of the lysosomal membrane proteome
Most of the currently known lysosomal membrane proteins, such as the sialic/glucuronic acid transporter sialin, the cysteine transporter cystinosin, the heparan sulfate α-glycosamine N-acetyltransferase, the putative cobalamin transporter LMBRD1 (LMBR1 domain) and the neuronal ceroid lipofuscinosis genes CLN3 and CLN7 have been identified by genetic mapping of disease-causing genes. More recently, proteomic approaches have been shown to be a proven method to reveal new components of the lysosomal membrane . Subproteomic analyses applying state-of-the-art mass spectrometric methodology for protein identification have provided important information about the protein composition of the lysosomal membrane, as reviewed in . In membranes purified from placental lysosomes, 58 proteins known to reside at least partially in the lysosomal membrane were identified . Some 86 additional proteins that were significantly enriched in the lysosomal membrane fraction were also observed. Among these, 12 novel proteins of unknown function were found. Among the proteins purified from the lysosomal membrane, 16 enzymes and transporters were also detected that had not been assigned to lysosomal membranes previously. The analysis of these novel membrane proteins will uncover completely new processes and functions associated with the lysosomal compartment.
This work was supported by the Deutsche Forschungsgemeinschaft.
We are grateful for the significant contributions made by Michael Schwake to experiments described in this paper.
Lysosomes in Health and Disease: A Biochemical Society Focused Meeting held at Charles Darwin House, London, U.K., 13–14 May 2010. Organized and Edited by Frances Platt (Oxford, U.K.) and Paul Pryor (York, U.K.).
Abbreviations: AMRF, action myoclonus–renal failure; β-GC, β-glucocerebrosidase; CMA, chaperone-mediated autrophagy; ER, endoplasmic reticulum; LAMP, lysosome-associated membrane protein; LDL, low-density lipoprotein; LIMP, lysosomal integral membrane protein
- © The Authors Journal compilation © 2010 Biochemical Society