The GTPase Arl13b participates in ciliary protein transport, but its contribution to intraflagellar transport (IFT), the main motor-based protein shuttle of cilia, remains largely unknown. Chlamydomonas arl13 mutant cilia were characterized by both abnormal reduction and accumulation of select membrane-associated proteins. With respect to the latter, a similar set of proteins including phospholipase D (PLD) also accumulated in BBSome-deficient cilia. IFT and BBSome traffic were apparently normal in arl13. However, transport of PLD, which in control cells moves by BBSome-dependent IFT, was impaired in arl13, causing PLD to accumulate in cilia. ARL13 only rarely and transiently traveled by IFT, indicating that it is not a co-migrating adapter securing PLD to IFT trains. In conclusion, the loss of Chlamydomonas ARL13 impedes BBSome-dependent protein transport, resulting in overlapping biochemical defects in arl13 and bbs mutant cilia.

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Two-color imaging of fluorescent protein-tagged IFT components indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B, and IFT dynein typically remain associated at the tip and anterograde trains convert directly into retrograde trains without disassembly but for possible splitting into strings of IFT complexes. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit preventing the build-up of IFT material in flagella.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of glycopeptides has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.