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
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
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
micrograph showing the core of a multivesicular endosomes within
a transformed mouse fibroblast.
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, 31053109, 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.
Red: expressed RMR-FLAG
Yellow denotes colocalization of the two integral membrane proteins.
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.
by Jeff Bocock
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.
Denotes colocalization of the two tetraspanins
by John Collette