Intracellular cobalamin (Cbl) utilization is critically dependent on its
efficient transit through the lysosome and subsequent delivery to cytosol
and mitochondria. We propose that age-related pathological processes
may inhibit lysosomal function and impair intracellular Cbl transport.
Purpose: To investigate alterations of intracellular [57Co]Cbl trafficking
in subcellular organelles when lysosome function is interrupted it is
essential to develop an optimal subcellular fractionation method to isolate
pure lysosomes, mitochondria and cytosol. Methods: Approximately 100
million human SH-SY5Y neurons or HT1080 fibroblasts were labelled
with [57Co]Cbl, homogenised using a ball-bearing homogeniser and the
lysates fractionated using an Optiprep gradient and reagent kits from
either Pierce or Sigma. [57Co] in each fraction was measured using
a gamma counter and subcellular fractions were probed by western
blotting. Results: Both protocols separated subcellular organelles to a
certain extent. The Pierce method seemed to be superior, separating pure
lysosomes from mitochondrial fractions without cytosol contamination.
SH-SY5Y [57Co]Cbl lysosome / mitochondria / cytosol distribution was 5.2
+ 0.4 / 13.2 + 0.6 / 81.6 + 0.8, respectively, and this was changed (all p <
0.01) to 54.2 + 1.9 / 7.0 + 0.9 / 35.5 + 2.7, respectively (all mean + SE, n =
3), when cells were treated for 48 h with 25 μM chloroquine (to increase
lysosomal pH). Similar results were obtained using HT1080 fibroblasts.
Conclusion: Development of subcellular fractionation methods provides
a useful tool for investigating intracellular Cbl trafficking. This method can
be adapted to study the impact of age- or pathology-related lysosomal
dysfunction on intracellular [57Co]Cbl transport.