Files
Abstract
Chlamydomonas PKD2 localizes into two distinct ciliary compartments (zones). In the distal cilium, PKD2 anchors mastigoneme in two rows. The distal PKD2 is stably connected to axoneme making it a stationary complex. The proximal cilium has more mobile PKD2 complex as it hops on-off from axoneme. Pull-down of detergent soluble PKD2 complex recognized a novel protein SIP, a single pass transmembrane protein. SIP has homology with the first transmembrane helix (TMH) of PKD2 and is part of both distal and proximal PKD2 complexes as sip knock-out has reduced ciliary PKD2 and no mastigoneme rows. Proximity labeling by PKD2-miniTurbo recognized a distally distinct protein, MST3, a 5-TMH protein like PKD2 with an incomplete PKD domain. The PKD domain of MST3 is predicted to be completed by SIP. In mst3 knockout strain distal PKD2 is largely absent. Another study via cryo-EM also discovered MST3 as the central backbone of mastigonemes. MST3 has a large extracellular domain adorned with numerous MST1 glycoproteins. Thus, MST3 is unique to the distal PKD2 complex. Chlamydomonas Genome scanning and domain predictions gave seven more gene hits whose gene products are 5-TMH proteins like MST3. All the seven proteins are predicted to form complex with SIP. Mass-spectrometry analysis showed that three out of the seven hits are ciliary proteins. To access, if the ciliary proteins are indeed a part of PKD2 interactome, endogenously tagged PKD2-mNGCRISPR strain has been established as a tool to generate knockouts of the respective proteins. One out of the three knock-outs, scv (scavenger), showed phenotypic defect in PKD2-mNG as it lacks the proximal PKD2. Endogenously tagged SCV-mNG localizes to the proximal cilia. Thus, SCV is a part of the proximal PKD2. Double knock-out mst3 scvPKD2-mNG reveals the abruption of both PKD2-mNG zones with only few randomly dotted PKD2-mNG moving via IFT (intraflagellar transport) indicating that MST3 and SCV are the distal and proximal zonal markers, respectively, for the specific PKD2 targeting. This study revealed a surprising heterogenicity of PKD2 complexes in cilia of Chlamydomonas. Thus, SIP, MST3 and SCV are established as novel interactors of PKD2 in Chlamydomonas.