Analysis of Protein Transport to Lysosomes

Abstract

Lysosomes are membrane-bound degradative organelles that are found in all higher eukaryotic cells. The limiting membranes of lysosomes contain a characteristic set of transmembrane glycoproteins that are transported to lysosomes by virtue of lysosomal targeting motifs found in the proteins' cytoplasmic tails (Bonifacino and Dell'angelica, 1999) The lumens of lysosomes contain an assortment of acidic hydrolases. These enzymes are synthesized in the endoplasmic reticulum (ER), where they become core-glycosylated. They are then transported into the Golgi complex, where they acquire a mannose-6-phosphate recognition tag in a two-step enzymatic process mediated by a phosphotransferase and an uncovering enzyme. The tag is then recognized by cation-dependent and cation-independent mannose-6-phoshate receptors in the trans-Golgi network (TGN). The resulting enzyme-receptor complexes are transported onward to endosomes, where the acidic pH induces dissociation of the enzymes from the receptors. The enzymes are then transported to lysosomes, while the receptors recycle back to the TGN to facilitate further rounds of transport. Failure of the receptors to recycle results in their misrouting to (and subsequent degradation in) lysosomes. The return of these receptors from endosomes to the TGN depends on specific amino acid residues (including specific cysteine residues that undergo reversible palmitoylation) in the cytoplasmic tails of receptor molecules. The final delivery of lysosomal enzymes to lysosomes has been proposed to occur by a fusion-fission process, referred to as "kiss and run," involving late endosomes and lysosomes (Luzio et al., 2003). BASIC PROTOCOL FRACTIONATION OF CELLS USING A SELF-FORMING PERCOLL DENSITY GRADIENT Sedimentation on self-forming Percoll gradients can be used to separate dense lysosomes from lower-density organelles such as endosomes, Golgi apparatus, plasma membrane, and ER. Separation is based on the sedimentation of particles to their isopycnic positioni.e., the position where the gradient density is equal to the density of the particle. The organelles are thus separated solely on the basis of differences in density, irrespective of their size. The gradient described in this protocol is a continuous gradient that forms

Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

“…In contrast, these complexes were highly enriched within both the DOC-soluble and DOC-insoluble fractions of ML-II Increased intracellular latent TGF-beta is localized within dense, detergent-insoluble fractions-To identify the intracellular location of accumulating latent TGFβ1, control and ML-II cellular homogenates were fractionated using Percoll gradients and the presence of latent TGFβ1 complexes probed by Western blot ( Figure 3A). Percoll gradient fractionation separates dense lysosomes and intermediate density organelles from other cellular organelles, including the endoplasmic reticulum and Golgi, providing a means to determine where intracellular latent TGFβ1 has accumulated within the secretory pathway [55,59]. The identity of individual fractions was confirmed with organelle-specific markers, including the lysosomal membrane protein LAMP-2, the ER protein ERp29 and the Golgi-associated protein GS-28.…”

A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

“…Fractions from the Percoll gradient were collected and then assayed individually for ␤-hexosaminidase activity as described. 20 The fractions with peak activity were pooled, transferred to 13 ϫ 51 mm thick-walled polycarbonate tubes, and spun at 4°C using a S100 AT4-542 rotor at 200 000g for 30 minutes to remove the Percoll. The supernatant was collected and used to digest immunotoxins.…”

Mechanical Forces Used for Cell Fractionation Can Create Hybrid Membrane Vesicles

“…Breaking the cell's membranes to retrieve smaller organelles, particles, proteins and nucleic acids is common practice. The study of processes such as pathogen invasion 1 , antigen presentation 2 - 4 , intracellular trafficking 3 , cellular signalling and dynamics, maturation and fusion of different compartments 4 , 5 and the analysis of protein complexes 6 demand organelle separation, determination of the organelle-specific markers and detection of foreign proteins or peptides on non-related membranes 7 . Imperative for these studies is that the technique applied does not influence the data retrieved.…”