Signals involved in protein intracellular sorting

“…Confusion appears to occur just after the articulation of a major conceptual advance
that served to greatly clarify a problem of exceptional importance.”- Ira Mellman, 1996.
What could be more fitting than the domain of protein trafficking to elucidate the above
statement made by one of the several pioneers in the field? Ever since the pioneering
groundwork laid down by Blobel and colleagues, emphasising protein translocation
across intracellular membranes, the field of protein trafficking has been a playground of
debates, dogma-reversals and rediscoveries. The possession of a valid cellular address
tag is the basic requirement for the delivery of a given protein at its intracellular
destination. However, the complexity involved in the foray of proteins from their site of
synthesis to their site of function is within the scope of no comprehensive treatise. In this
thesis, the work done on two individual transport steps of two different proteins has been
summarised.
In the first part of this thesis, the trafficking of the cation-dependent (CD-) mannose 6-
phosphate receptor (MPR) has been studied. The CD-MPR cycles between the TGN and
the plasma membrane, through the early and late endosomal compartments. It performs
the important function of transport of lysosomal enzymes to lysosomes, a process which
ensures the correct biogenesis of lysosomes. However, it is important that the receptor
itself be excluded from lysosomes and safely retrieved to the TGN from late endosomes
in order to avoid degradation in lysosomes. This is essential to ensure that the CD-MPR
is available to support several rounds of lysosomal enzyme transport. This retrieval step
has been shown to depend on a pair of aromatic residues F18W19 in the cytoplasmic tail
of the receptor. Mutation of the residues to alanines has been shown to result in massive
mislocalisation of the CD-MPR in lysosomes, the W19 residue being more crucial to
this function and the F18 residue playing a contributory role. The retrieval has also been
shown to take place in a Rab9 dependent manner using the cytosolic adaptor protein
TIP47 (Tail Interacting Protein of 47 kDa). TIP47 specifically interacts with the diaromatic
motif to effect this transport step. In this study, we demonstrated a strict
requirement for di-aromaticity at the positions 18 and 19 of the cytosolic tail of the CDMPR
both for correct intracellular sorting in vivo and optimal TIP47 interaction in vitro,
thus demonstrating the significance of the di-aromatic motif in endosomal sorting and establishing the highly specific nature of this interaction. This also established a
paradigm for the CD-MPR as a representative member of a generic family of diaromatic
motif containing proteins.
The second part of this thesis deals with the trafficking of the human mannose 6-
phosphate uncovering enzyme (UCE). The recognition of the mannose 6-phosphate tag
on lysosomal enzymes by the MPRs is facilitated by UCE which exposes the recognition
signal on the lysosomal enzymes in a two-step enzymatic reaction: the first starts in the
cis-Golgi and is mediated by a phosphotransferase and the second, mediated by UCE,
occurs in the TGN. At steady state, UCE is mostly localised to the TGN and it cycles
between the TGN and the plasma membrane. It is rapidly internalised from the surface
in a clathrin dependent endocytic pathway and the internalisation has been shown to be
mediated by a critical tyrosine-488 residue in its cytoplasmic tail. The transmembrane
domain and first 11 residues of the cytoplasmic tail of UCE have been shown to be
involved in its TGN retention. In this study, we identified the residues involved in TGN
exit of UCE using a combination of biochemical and confocal immunofluorescence
methods. Using a high dimensional neural network capable of identifying differences
between images not visible to the eye, we determined that the residues 492QEMN were
involved in TGN exit of UCE. The same method was also used to analyse the individual
contribution of each amino acid in the sequence and it was found that residue Q492 is the
most important to the exit function while residues M494 and N495 also contribute. The
identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the
trafficking pathway of uncovering enzyme, a crucial player in lysosomal biogenesis.
With these two analyses, we contributed to a better understanding of signal sequences
involved in intracellular protein trafficking of two related proteins both involved in
lysosomal biogenesis.