Protein Targeting

    A newly synthesized protein must be recognized and modified by a variety of proteins in order to reach its final cellular destination in the correct biosynthetic form. Recognition of phosphorylated mannose residues by cellular receptors normally leads lysosomal proteases to be efficiently targeted to lysosomes, where they participate in the cooperative mediation of terminal degradation of endocytosed and endogenous proteins.

    Under certain physiological conditions, lysosomal protease targeting is altered so that newly synthesized enzymes are secreted rather than targeted to lysosomes. In many tumors, the synthesis and secretion of a particular lysosomal protease is increased relative to normal cells in the surrounding tissue. The secreted protease is thought to aid tumor cell metastasis by participating in the degradation of the extracellular matrix through which the cells must migrate in order to establish secondary tumors.

     We are characterizing a mannose phosphate-independent or alternate targeting pathway that leads to the accumulation of a lysosomal proenzyme in multivesicular endosomes, vesicles that can secrete their contents in response to specific signals. The targeting of one protease to two distinct cellular sites, lysosomes and multivesicular endosomes, suggests the existence of cellular proteins, in addition to mannose phosphate receptors, which specifically recognize and target the protease within cells. We are studying four proteins localized to multivesicular endosomes that may potentially modulate targeting of the lysosomal cysteine protease cathepsin L.

Electron micrograph showing the core of a multivesicular endosomes within a transformed mouse fibroblast.

Modified from: Traffic 3:147-150, 2002

REFERENCE: Collette, J., Ulku, A. S., Der, C. J., Jones, A.'S., ERICKSON, A. H. (2004) "Enhanced cathepsin L expression is mediated by different ras effector pathways in fibroblasts and epithelial cells," Intl. J. Cancer 112, 190-199.


    RMR/Rnf13 is an endosomal membrane protein of unknown function that has a protease-associated or PA luminal domain and a RING finger domain in its cytoplasmic tail. The plant homologue is localized to protein storage bodies (J. Cell Biol. 150:755-70, 2000), similar to mammalian multivesicular endosomes, while the chicken homologue has been identified in nuclei (Proc. Natl. Acad. Sci. :93, 3105–3109, 1996). We are characterizing the biosynthesis, targeting and function of this new protein that may, in analogy to the plant protein, bind a lysosomal cysteine protease. The challenge is to determine whether this one protein actually serves both as a receptor and as a transcription factor.

    We find the RMR protein is an integral membrane protein that shows a punctate distribution characteristic of endosomes in mammalian cells. The protein partially colocalizes with tetraspanin CD63, a marker protein for multivesicular bodies. We are assaying the protein for ubiquitin ligase activity, a common function of RING fingers, preparing mutants of the protein to determine the role various protein determinants play in the targeting of RMR to multivesicular endosomes, and assaying for proteins that interact with RMR.

Green: endogenous CD63

Red: expressed RMR-FLAG

Yellow denotes colocalization of the two integral membrane proteins.

Micrograph by Jeff Bocock

REFERENCE: Jiang, L., ERICKSON, A. H. and Rogers, J. C. (2002) "Multivesicular bodies: a mechanism to package lytic and storage functions in one organelle?," Trends in Cell Biol.12, 362-367.


    Testican-1 is an extracellular matrix protein that is unique because it contains a series of domains which each potentially inhibit a different protease family. With Dr. Cora-Jean Edgell, the UNC Department of Physiology, who first cloned and characterized this proteoglycan, we established that testican can inhibit cathepsin L, a lysosomal protease which is massively secreted by transformed mouse fibroblasts. By immunofluorescence microscopy, we find that testican is not only localized outside cells, as expected for a proteoglycan, but is also found intracellularly in a punctate pattern indicative of localization in vesicles. From electron microscopy studies we know that procathepsin L is stable in endosomes which have an internal matrix likely formed by a polymer such as a proteoglycan. In collaboration with Dr. Edgell, we are determining whether the lysosomal enzyme and its inhibitor are localized in the same intracellular vesicles. We are also assaying whether secreted cathepsin L cleaves testican to release the cathepsin L inhibitory thyropin domain to the serum, enabling it to regulate protease activity at a distant site.

Green: testican-1

Micrograph by Jeff Bocock

REFERENCE: Bocock, J. P., Edgell, C.-J. S., Marr, H. S., and ERICKSON, A. H. (2003) "Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L," Eur. J. Biochem. 270, 4008-4015.


     Tetraspanins, proteins that cross a membrane four times, are frequently localized in multivesicular endosomes. CD63 is found in multivesicular bodies with procathepsin L (electron micrograph above) and with another tetraspanin, CD82. We are assaying for colocalization of the tetraspanins with cathepsin L in multivesicular endosomes by immunofluorescence, sucrose gradient cell fractionation, and by coimmunoprecipitation.

Red: CD82

Green: CD63

Yellow: Denotes colocalization of the two tetraspanins

Photographs by John Collette