Several functions have been suggested for lysosomal membrane proteins, such as sequestration of numerous acid hydrolases, maintenance of an acidic intralysosomal environment, transport of degradation products out of lysosomes, and specific interaction and fusion events between lysosomes and other organelles (Fukuda, 1991
; Peters and von Figura, 1994
; Eskelinen et al., 2003
). LAMP-1 and -2, the two most abundant lysosomal membrane proteins, have been estimated to contribute to ~50% of all proteins of this membrane (Hunziker et al., 1996
). The presence of LAMP molecules is one of the major definitions of the lysosomal compartment (Kornfeld and Mellman, 1989
). What then was the consequence of total absence of both major lysosomal membrane proteins on lysosomal structure and function? Surprisingly, we observed that typical lysosomes, as defined by ultrastructural appearance, and capacity to take up fluid phase markers and degrade proteins, still existed in LAMP-1/LAMP-2 double-deficient MEFs. It is likely that other lysosomal membrane proteins including LIMP 1 and LIMP-2/LGP-85 were able to take over the protective function against lysosomal hydrolases.
However, the generation of LAMP-1/LAMP-2 double-deficient embryos described in this study demonstrated that these proteins fulfill at least partially overlapping functions in vivo. The tight functional association of the proteins is also highlighted by the observation that mice with only one functional LAMP allele were more susceptible to early postnatal mortality. Although no gross impairment of organ development was encountered, LAMP-1/LAMP-2 double-deficient embryos were characterized by a massive accumulation of autophagic vacuoles in many tissues, especially endothelial cells. It is possible that further embryonic development beyond embryonic day E15 was prevented due to these cellular alterations and impaired delivery of nutrients.
We showed earlier that autophagic vacuoles accumulate in LAMP-2 single-deficient liver tissue and isolated hepatocytes (Tanaka et al., 2000
; Eskelinen et al., 2002a
). This accumulation could be attributed to a decreased lysosomal degradation of long-lived proteins. In this study we observed an accumulation of late autophagic vacuoles in the LAMP double-deficient cells. Surprisingly, in LAMP-1/LAMP-2 double-deficient MEFs the protein degradation rates under all conditions tested were similar to control cells, suggesting that the accumulation of autophagic vacuoles was not due to retarded protein degradation. Thus, the increased accumulation could be due to impaired degradation of other macromolecules including lipids or possibly to defective export of degradation products from autophagic vacuoles to the cytoplasm. It is also possible that in the double-deficient cells, retarded protein degradation per autophagic vacuole was compensated by the increased amount of vacuoles, and therefore we did not see a difference in protein degradation rates between control cells and the double-deficient cells.
One of the three LAMP-2 isoforms, LAMP-2a, has been suggested to be the receptor for the chaperone-mediated autophagy, a selective uptake and degradation of cytosolic proteins by lysosomes (Cuervo and Dice, 1996
). This pathway was shown to be active in confluent lung fibroblasts and Chinese hamster ovary cells cultured in serum-free medium (Cuervo and Dice, 1998
). In this study we analyzed MEFs deficient in LAMP-2, LAMP-1, or both. In LAMP-2-deficient cells all LAMP-2 isoforms including LAMP-2a were disrupted. MEF cells deficient in LAMP-2 or both LAMP-1 and -2 did not show changes in the lysosomal protein degradation rates under six different conditions. These results suggested that deficiency of LAMP-2 or both LAMP-2 and LAMP-1 did not alter lysosomal protein degradation rates even in confluent cells under prolonged (28 h) serum starvation, a condition where chaperone-mediated autophagy is supposed to be more active. Thus, it is possible that LAMP-2 is not the only receptor in chaperone-mediated autophagy or that the pathway is not active in mouse embryonic fibroblasts, like in other cells. However, the latter alternative appears unlikely because the proteolytic rates measured in our MEFs resembled those of human fibroblasts, where the pathway is known to be active. Another possibility is that other proteolytic pathways compensate for the defective activity of chaperone-mediated autophagy in LAMP-2-deficient mouse fibroblasts. Although our experiments with various inhibitors excluded this for macroautophagy, proteasomal degradation, and nonlysosomal proteolytic pathways different from proteasomes, the possibility still exists that a lysosomal pathway that is different from macroautophagy (such as microautophagy) is activated to compensate for the deficiency in chaperone-mediated autophagy.
We observed an altered subcellular distribution of MPR300 in LAMP double-deficient MEFs. Despite this, cathepsin D delivery to the lysosomal compartment and uptake of arylsulfatase A, a mannose-6-phosphate-containing ligand, from the culture medium were not affected (unpublished data). This was in contrast to LAMP-2 single-deficient hepatocytes where we observed that lysosomal biogenesis, MPR46 expression levels (Eskelinen et al., 2002b
) and MPR300 function in endocytosis (unpublished data) were affected. This may be explained by different expression levels of LAMP proteins and different requirements for lysosomal functions in embryonic fibroblasts and adult hepatocytes.
Intriguingly, we found altered lipid localization in LAMP-1/LAMP-2 double-deficient and LAMP-2 single-deficient MEFs. Prominent storage of unesterified cholesterol in endo/lysosomal compartments of LAMP-1/LAMP-2 double-deficient cells was observed. Transfection with wild-type LAMP-2a constructs reversed the cholesterol storage phenotype, whereas LAMP-1 transfection had a much smaller effect, which demonstrated the major importance of LAMP-2 for the cholesterol metabolism. The decreased amount of Nile Red-stained lipid droplets in the double-deficient MEFs also indicated that lipid metabolism was altered. Recent studies have demonstrated that lipid droplets are not passive storage structures but have an active role in lipid metabolism (Liu et al., 2004
The pattern of cholesterol storage in LAMP-1/LAMP-2-deficient cells resembled that in fibroblasts from Niemann-Pick type C patients (Garver and Heidenreich, 2002
). Similar to LAMP double-deficient cells, these cells accumulated unesterified cholesterol in late endosomal/lysosomal vesicles. Mutations in NPC1 or in NPC2 have been described to be responsible for the cholesterol storage phenotype (Garver and Heidenreich, 2002
). It has been reported that after transfection of NPC1 to cells deficient in this protein, the protein first localized to the limiting membranes of cholesterol-loaded endo/lysosomes. After cholesterol had been cleared from endo/lysosomes, the localization of the reexpressed NPC1 changed to small late endosomal filipin-negative vesicles, which was identical to the localization of NPC1 in control cells (Zhang et al., 2001
). We observed that in LAMP double-deficient cells NPC1 localized in the cholesterol-loaded endo/lysosomes, whereas in control cells NPC1 localized in small filipin-negative vesicles. Thus, it is likely that in the double-deficient cells NPC1 was recruited to the correct localization for cholesterol clearance, but it was not able to fulfill this function. The altered distribution of NPC1-GFP could be reversed by parallel expression of NPC1-GFP and LAMP2, indicating that LAMP-2 directly or indirectly influenced the trafficking or function of NPC1. Similar to NPC1, rab7 also localized in the cholesterol-loaded endosomes in the LAMP double-deficient cells. It has been reported that cholesterol accumulation in the lysosomal compartment increases the amount of membrane-associated rab7 (Lebrand et al., 2002
) and this was suggested to interfere with the rab7 function. Thus, it is possible that at least some of the alterations observed in the LAMP double-deficient cells, such as altered localization of lysosomal vesicles in the peripheral cytoplasm, were due to impaired function of rab7. Further studies are however necessary to clarify the mechanistic reasons for the accumulation or cholesterol and decreased amount of lipid droplets in MEFs deficient of LAMP-1 and -2.
In conclusion, 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 indicated that these two major lysosomal membrane proteins share common functions in vivo. However, LAMP-2 seemed to have more specific functions because LAMP-2 single deficiency had more severe consequences than LAMP-1 single deficiency. In embryonic fibroblasts, mutual disruption of both LAMPs was associated with an increased accumulation of autophagic vacuoles, altered lysosomal distribution and appearance, and disturbed cholesterol metabolism, whereas protein degradation rates were not affected. These results clearly show that the LAMP proteins fulfill functions far beyond the initially suggested roles in maintaining the structural integrity of the lysosomal compartment.