Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells

FWN – Publicaties zonder aanstelling Universiteit Leide

Mannose 6-phosphate independent targeting of cathepsin D to lysosomes in HepG2 cells

FWN – Publicaties zonder aanstelling Universiteit Leide

Imaging and imagination: understanding the endo-lysosomal system

Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today

Differential effects of brefeldin A on transport of secretory and lysosomal proteins

Brefeldin A (BFA) rapidly blocks anterograde exocytotic transport through the Golgi complex. Sustained retrograde traffic induced by brefeldin A causes redistribution of constituents of the Golgi, but not the trans-Golgi network (TGN), to the endoplasmic reticulum (ER). In the present study on HepG2 cells, we have observed a differential effect of BFA on transport from the TGN of two soluble proteins: α1-antitrypsin as a representative of secretory proteins and cathepsin D as a prototype of lysosomal enzymes. The Golgi complex of HepG2 cells is sensitive to BFA, as within minutes after its addition nearly all activity of three resident Golgi enzymes was recovered in the ER as monitored by cell fractionation on sucrose density gradients. In accordance with this, 'high mannose'-glycosylated α1-antitrypsin was retained in or transported back to the ER. 'Complex'-glycosylated α1- antitrypsin was neither secreted into the medium nor transported back to the ER. Most of it was retained in vesicles with the buoyant density of Golgi. These vesicles contained the fluid phase endocytotic marker horseradish peroxidase when this was added to the culture medium prior to the BFA, suggesting that the vesicles derived from the TGN. After BFA addition, the compartment became inaccessible to endocytosed horseradish peroxidase. In contrast to blocking transport of complex α1-antitrypsin, BFA did not affect processing of newly synthesized complex-glycosylated procathepsin D (53 kDa) to the mature 31-kDa form. Neither did it interfere with processing of endocytosed procathepsin D. That the mature cathepsin D had indeed reached the lysosomes was verified by Percoll density gradient fractionation. In conclusion, in HepG2 cells, BFA induces two blocks in the secretory pathway: one at the level of the ER-Golgi juncture and the other in the TGN. In contrast, transport from the Golgi complex to the lysosomes and from the plasma membrane to the lysosomes continued

Functional activity of the reduced folate carrier in KB, MA104 and IGROV-I cells expressing folate-binding protein

The role of a membrane-associated folate binding protein (mFBP) in transport of folate analogues was investigated in three epithelial cell lines that were grown in high folate medium and folate-conditioned medium and express different levels of mFBP: human nasopharyngeal KB cells, monkey kidney MA104 cells, and IGROV-I ovarian carcinoma cells. Folate analogues were selected for which mFBP exhibits a low affinity, i.e., methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folic acid, i.e., N-(5[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl(-N-m ethylamino]-2-theonyl)-L-glutamic acid (ZD1694), N10-propargyl-5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies with IGROV-I and MA104 cells demonstrated a lack of correlation between the affinity of mFBP for the antifolate drugs and their sensitivity profile; both cell lines were highly sensitive to growth inhibition by MTX, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were insensitive for CB3717. The same drug sensitivity profile was observed for KB cells, with the exception that these cells were also sensitive to growth inhibition by CB3717 but only in folate-conditioned medium. This overall drug sensitivity profile appeared to correlate with the differential efficiency of drug transport via the "classical" reduced folate/MTX carrier (RFC), rather than by mFBP. Characteristics that further supported functional RFC activity in KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prevented by the reduced folate leucovorin rather than by folic acid; (b) rates for uptake of [3H]10-EdAM were 2-4-fold higher than for [3H]MTX at 1 microM extracellular concentrations and coincided with the affinity of the RFC for these drugs, rather than those of the mFBP; (c) uptake of [3H]10-EdAM and [3H]leucovorin was markedly inhibited by leucovorin and 10-EdAM, respectively, or by an N-hydroxysuccinimide ester of MTX (irreversibly labeling RFC) but only to a minor extent by folic acid or an N-hydroxysuccinimide ester of folic acid (irreversibly labeling mFBP); and, finally, (d) labeling with an N-hydroxysuccinimide ester of [3H]MTX identified a protein with a molecular weight within the range of that reported for the RFC in human leukemic cells. Altogether, these results indicate that both RFC and mFBP are coexpressed in three epithelial cell lines and that RFC is the preferential route of entry for antifolate compounds, even when mFBP is expressed to very high levels