The exocyst complex and intracellular vesicles mediate soluble protein trafficking to the primary cilium.

The efficient transport of proteins into the primary cilium is a crucial step for many signaling pathways. Dysfunction of this process can lead to the disruption of signaling cascades or cilium assembly, resulting in developmental disorders and cancer. Previous studies on the protein delivery to the cilium were mostly focused on the membrane-embedded receptors. In contrast, how soluble proteins are delivered into the cilium is poorly understood. In our work, we identify the exocyst complex as a key player in the ciliary trafficking of soluble Gli transcription factors. In line with the known function of the exocyst in intracellular vesicle transport, we demonstrate that soluble proteins, including Gli2/3 and Lkb1, can use the endosome recycling machinery for their delivery to the primary cilium. Finally, we identify GTPases: Rab14, Rab18, Rab23, and Arf4 that are involved in vesicle-mediated Gli protein ciliary trafficking. Our data pave the way for a better understanding of ciliary transport and uncover transport mechanisms inside the cell.

MRCKα/ß positively regulates Gli protein activity.

Posttranslational modifications (PTMs) are key regulatory events for the majority of signaling pathways. Transcription factors are often phosphorylated on multiple residues, which regulates their trafficking, stability, or transcriptional activity. Gli proteins, transcription factors that respond to the Hedgehog pathway, are regulated by phosphorylation, but the sites and the kinases involved have been only partially described. We identified three novel kinases: MRCKα, MRCKß, and MAP4K5 which physically interact with Gli proteins and directly phosphorylate Gli2 on multiple sites. We established that MRCKα/ß kinases regulate Gli proteins, which impacts the transcriptional output of the Hedgehog pathway. We showed that double knockout of MRCKα/ß affects Gli2 ciliary and nuclear localization and reduces Gli2 binding to the Gli1 promoter. Our research fills a critical gap in our understanding of the regulation of Gli proteins by describing their activation mechanisms through phosphorylation.

The role of plastidial glucose-6-phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.