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.
Pisoni
R L
RL
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109-2029.
Acker
T L
TL
Lisowski
K M
KM
Lemons
R M
RM
Thoene
J G
JG
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