Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits.

The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human.