Interakcija proteina IqgC i Rab5A u amebi Dictyostelium discoideum

master's thesis

• Title (english): Interaction between Dictyostelium discoideum proteins IqgC and Rab5A
• Author: Tamara Ćutić
• Mentor: Vedrana Filić Mileta (mentor)
• Mentor: Ivana Ivančić Baće (sumentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-02-22, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Protein IqgC iz amebe Dictyostelium discoideum sudjeluje u makropinocitozi, neselektivnom
unosu izvanstanične tekućine, kao negativni regulator proteina RasG. Protein RasG je mala
GTPaza koju protein IqgC inaktivira poticanjem hidrolize vezanog GTP-a, odnosno IqgC
djeluje kao njen GAP (engl. GTPase activating protein). Proteini IqgC i RasG su tijekom
makropinocitoze smješteni na makropinocitotsku čašicu i novonastali makropinosom, ali IqgC
ostaje na makropinosomu i nakon što je RasG disocirao. To sugerira da IqgC na
makropinosomu zadržava interakcija s drugim proteinskim i/ili fosfolipidnim partnerom. Iz
objavljenog interaktoma IqgC probrani su potencijalni proteini kandidati na temelju njihove
uloge u sazrijevanju ranog makropinosoma te su preliminarni pokusi pokazali da se mala
GTPaza Rab5A nalazi u kompleksu s IqgC. Cilj ovog rada bio je istražiti je li interakcija
proteina IqgC i Rab5A izravna te usporediti prostorno-vremenske raspodjele ova dva proteina
tijekom makropinocitoze. Metodom GST-pull-down potvrdili smo da se pročišćeni protein IqgC
izravno veže na Rab5A. Koristeći konfokalnu mikroskopiju, pokazali smo da IqgC i Rab5A
kolokaliziraju na makropinosomima, pri čemu IqgC disocira s vezikule znatno prije nego što
Rab5A dosegne maksimalni intenzitet. Za razjašnjavanje fiziološke uloge ove interakcije
potrebna su daljnja istraživanja.

Abstract (english)

Dictyostelium discoideum IqgC protein interacts with the small GTPase RasG and promotes its
GTP-hydrolytic activity, i.e. acts as its GAP (GTPase activating protein). By suppressing Ras
activity, it negatively regulates macropinocytosis, the bulk uptake of extracellular fluid. During
macropinocytosis, both IqgC and RasG localize to macropinocytic cup and newly formed
macropinosome, but IqgC remains on the nascent macropinosome even after RasG dissociated.
This suggests that another protein and/or phospholipid interactor engages with IqgC on the
macropinosome. Few potential protein candidates have been selected from the published IqgC
interactome, based on their known functions during early endosome maturation. Among them
was the small GTPase Rab5A that was shown to exist in a complex with IqgC. In this work, we
aimed to explore whether this interaction between IqgC and Rab5A is direct and to compare
spatio-temporal distributions of these proteins during macropinocytosis. We performed a GSTRab5A-pull-down assay and confirmed that purified IqgC binds directly to Rab5A. Using
confocal microscopy, we demonstrated colocalization of Rab5A and IqgC on macropinosomes
with IqgC leaving the vesicle well before Rab5A reached maximal intensity. Deciphering the
physiological significance of this interaction requires further investigation.

• Keywords: IQGAP; RasGAP; GTPaza; endocitoza; makropinocitoza
• Keywords (english): IQGAP; RasGAP; GTPase; endocytosis; macropinocytosis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:122699
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-08-22
• Created on: 2022-03-23 13:16:39