The Small Interactor of PKD2 protein promotes the assembly and ciliary entry of the Chlamydomonas PKD2–mastigoneme complexes

Abstract

In Chlamydomonas, the channel PKD2 is primarily present in the distal region of cilia, where it is attached to the axoneme and mastigonemes, extracellular polymers of MST1. In a smaller, proximal ciliary region, PKD2 is more mobile and lacks mastigonemes. We show that the PKD2 regions are established early during ciliogenesis and increase proportionally in length as cilia elongate. In chimeric zygotes, tagged PKD2 rapidly entered the proximal region of PKD2-deficient cilia whereas assembly of the distal region was hindered, suggesting that axonemal binding of PKD2 requires de novo assembly of cilia. We identified Small Interactor of PKD2 (SIP), a PKD2-related, single-pass transmembrane protein, as part of the PKD2-mastigoneme complex. In sip mutants, stability and proteolytic processing of PKD2 in the cell body were reduced and PKD2-mastigoneme complexes were absent from the cilia. Like the pkd2 and mst1 mutants, sip swam with reduced velocity. Cilia of the pkd2 mutant beat with an increase frequency but were less efficient in moving the cells, suggesting a structural role for the PKD2-SIP-mastigoneme complex in increasing the effective surface of Chlamydomonas cilia.

Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices

“… 47 Using negative-stain electron microscopy, we first confirmed that the mst1, sip , and pkd2 mutants lack mastigonemes ( Figure S5A ), as previously reported. 16 , 19 We next examined four different mst3 mutants and observed that all lack mastigonemes ( Figures S5B and S5C ), consistent with the proposed central role of MST3 in mastigoneme formation. The mst3 mutant strains also displayed a modest (~15%) but statistically significant reduction in swimming velocity compared with wild-type (WT) strain ( Figure S6A ) that is similar to the reduction reported in some studies with mst1, pkd2 , and sip mutants.…”

“…A role for SIP in anchoring mastigonemes to PKD2 in the membrane explains the lack of mastigonemes in sip mutants. 19 Taken together, we propose that PKD2, MST3, and SIP form a complex in the ciliary membrane with a 3:1 stoichiometry between PKD2 and the MST3:SIP complex.…”

“…Remarkably, this exact missing region is encoded by SIP, a small protein recently identified to copurify with C. reinhardtii PKD2. 19 We therefore speculated that SIP could bind MST3 to complete the PKD fold. Consistent with this idea, an AlphaFold2 multimer prediction of the MST3:SIP complex bears a striking resemblance to PKD2 ( Figures 6C – 6E ).…”

“…The mst3 mutant strains also displayed a modest (~15%) but statistically significant reduction in swimming velocity compared with wild-type (WT) strain ( Figure S6A ) that is similar to the reduction reported in some studies with mst1, pkd2 , and sip mutants. 16 , 19 …”

“… 16 The exact function of mastigonemes is unknown. A proposed role in increasing the efficiency of the ciliary beat 13 , 16 , 18 , 19 has not been observed in all studies. 15 A potential function in adhesion during mating has also been refuted.…”