Intracellular pathway followed by invertase endocytosed by rat liver
Authors: JADOT Michel , MISQUITH Sandra , DUBOIS Franz , WATTIAUX‐DE CONINCK Simone , WATTIAUX Robert 
Published: March 2005

Yeast invertase, when injected into rats, is endocytosed by the liver, mainly by sinusoidal cells. The work reported here aims at investigating the organelles involved in the intracellular journey of this protein. Experiments were performed on rats injected with 125I‐invertase (25 μg/100 g body wt) and killed at various times after injection.

Homogenates were fractioned by differential centrifugation, according to de Duve, Pressman, Gianetto, Wattiaux and Appelmans [(1955) Biochem. J. 63, 604–617]. Early after injection the radioactivity was recovered mainly in the microsomal fraction P; later it was found in the mitochondrial fractions (ML). At all times a peak of relative specific activity was observed in the light mitochondrial fraction L.

After isopycnic centrifugation in a sucrose gradient, structures bearing 125I‐invertase, present in P, exhibited a relatively flattened distribution with a density of around 1.17 g/ml, relatively similar to that of alkaline phosphodiesterase a plasma membrane marker. The organelles located in ML were endowed with a more homogeneous distribution, their median equilibrium density increasing up to 30 min after injection (1.20 g/ml → 1.23 g/ml); with time the radioactivity distribution became more closely related to the distribution of arylsulfatase, a lysosomal enzyme.

ML fractions, isolated 10 min and 180 min after 125I‐invertase injection, were subjected to isopycnic centrifugation in Percoll gradient with, as solvent, 0.25 M, 0.5 M and 0.75 M sucrose. The change of density of the particles bearing 125I‐invertase, as a function of the sucrose concentration, paralleled the change of density of the lysosomes as ascertained by the behaviour of arylsulfatase.

The distribution of radioactivity and arylsulfatase in a sucrose gradient was estalished after isopycnic centrifugation of the ML fraction of rats injected with 125I‐invertase, the animals having received or not an injection of 900 μg/100 g body weight of unlabelled invertase 15 h before killing. In agreement with our previous results, a shift towards higher densities of about 25% or arylsulfatase takes place in rats pretreated with unlabelled invertase. At 10 min, invertase preinjection did not change the radioactivity distribution curve. Later, it caused a progressive shift of the distribution towards higher‐density regions of the gradient where the arylsulfatase, which had been shifted, was located.

The release by glycyl‐l‐phenylalanine 2‐naphthylamide (Gly‐l‐Phe‐2‐NH‐Nap), a compound that disrupts the lysosomal membrane, of the radioactivity associated with P and ML was investigated. Gly‐l‐Phe‐2‐NH‐Nap caused a release of 20% radioactivity present in P, which did not change with injection time. The percentage of radioactivity located in ML that could be released by Gly‐l‐Phe‐2‐NH‐Nap increased with time following injection until it reached 85% of that which could be released by Triton X‐100.

These results can be best explained by supposing that invertase endocytosed by liver sinusoidal cells travels successively through three intracellular compartments: pinocytic vesicles, endosomes and ‘transfer’ lysosomes before reaching lysosomes in which it finally accumulates.