Molecular insights into the transport lectin function of ERGIC-53

Appenzeller-Herzog, Christian. Molecular insights into the transport lectin function of ERGIC-53. 2004, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_6816

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Secretion of proteins is an essential function of eukaryotic cells. The secretory proteins’
journey along the organelles of the exocytic pathway is initiated by the exit from the
endoplasmic reticulum (ER), which defines a major rate-limiting step for protein secretion.
ER-exit is subject to tight quality control. Selective, receptor-mediated cargo capture is one of
the mechanisms thought to contribute to the elaborate proof-reading system of the ER.
The recycling mannose lectin ERGIC-53 operates as an ER-export receptor of a subset
of secretory glycoproteins. The required signals for this transport step, however, remain
poorly described. Experiments in this thesis show that ERGIC-53 assisted ER-exit of
procathepsin Z depends on a novel transport motif that is composed of a high-mannose type
oligosaccharide and a peptide β-hairpin loop. Deletion of either determinant compromises
ERGIC-53 association and slows procathepsin Z transport. An equivalent
carbohydrate/hairpin structure is identified in cathepsin C, another cargo of ERGIC-53,
reflecting the general nature of this ER-export signal. Further experiments reveal that the Nglycans
of loop-deficient procathepsin Z become efficiently mannose 6-phosphorylated, but
undergo increased carbohydrate processing in the Golgi including complex glycosylation.
Strikingly, cathepsin Z lacking the peptide loop is not targeted to its normal destination, the
lysosome, suggesting that it lacks the correct carbohydrate signal for lysosomal delivery. The
presented data describe the first ER-exit signal on a secretory protein and establish an
unexpected link between lectin-mediated export from the ER and post-Golgi sorting.
This thesis also provides the molecular basis for ERGIC-53/cargo dissociation in the
ERGIC. In vitro mannose binding experiments reveal that the lectin only displays its full
activity at pH 7.4 – the pH of the ER – but not at slightly lower pH. The acid-sensitivity is
modulated by the calcium concentration indicating a molecular link between pH-sensing and
calcium complexation. This link is spotted by the identification of His178 that is conserved
throughout the family of animal L-type lectins and – in its deprotonated form – binds a
calcium ion in the carbohydrate recognition domain (CRD) of ERGIC-53. pH-induced
inactivation of ERGIC-53 is also shown in cell culture. Glycoprotein binding is inhibited, if
the ER is acidified, and the kinetics of glycoprotein dissociation are slowed, if the ERGIC is
neutralized. The results establish the ERGIC as the earliest acid compartment of the secretory
pathway and suggest that pH-induced glycoprotein dissociation may be backed by a
mechanism that maintains lower levels of free calcium in the ERGIC. The organelles of the secretory pathway operate as intracellular calcium stores. High
concentrations of calcium have been measured in the lumen of the ER and the Golgi, but the
calcium concentration in the ERGIC is not known. Therefore, a strategy was developed to
quantitatively assess the free calcium concentration of the ERGIC in vivo using the green
fluorescent protein-based calcium-indicator yellow cameleon. Targeting of the indicator to the
ERGIC is achieved by fusing it to an inert variant of the ER-Golgi SNARE Sec22b. The
fusion protein dynamically localizes to the ERGIC without disturbing the function of the
endogenous SNARE machinery. It will in the future provide a valuable tool for calcium
measurements in the ERGIC.
Advisors:Hauri, Hans-Peter
Committee Members:Spiess, Martin
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Pharmacology/Neurobiology (Hauri)
UniBasel Contributors:Hauri, Hans-Peter and Spiess, Martin
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6816
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:121
Identification Number:
Last Modified:05 Apr 2018 17:32
Deposited On:13 Feb 2009 15:28

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