A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis.
Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 microM [35S]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37 degrees C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Q10 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [35S]L-cysteine: ala, ser, pro, val, gly, homocysteine, D- or L-penicillamine, arg, asp, or leu. Similarly, D-cysteine and beta-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and alpha-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an alpha-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteine-specific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109-2029.
R L
RL
Pisoni
T L
TL
Acker
K M
KM
Lisowski
R M
RM
Lemons
J G
JG
Thoene
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism / Q000493:pharmacokinetics* / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008247:Lysosomes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D010447:Peptide Hydrolases; D003545:Cysteine