SummaryNutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signaling motivated us to explore the possible functional interactions between this signaling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signaling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. While abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie the basis of some common ciliopathies.

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Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freezefracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles--trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (75 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18~ while in cells grown at the nonpermissive temperature (27~ the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses," also found in wild-type and in nd9 (18~ cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.All freeze-fractured natural membranes studied so far display a smooth background that probably represents regions of lipid bilayer (5, 7), on which "particles" corresponding to proteins or lipoprotein aggregates (9, 20) are usually found. These particles generally appear randomly distributed but various types of organized particle arrays have been described: for instance, the arrays involved in 126

Synchronous secretion of all available mature mucocysts was induced in late log phase cultures of Tetrahymena thermophilia (B III) by the local anaesthetic dibucaine. No assembled fusion rosettes were seen within the plasma membrane after release until 2-3 hrs of regrowth, thus proving that the rosettes are not permanent sites within the plasma membrane but have to be reassembled each time for a new fusion event to occur. Concomitant with the reappearance of assembled fusion rosettes, the cell cytoplasm fills up with precursors of new mucocysts thus linking the two events together.

A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a uniceilular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutate activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specifi to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins-were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.Previously we discovered a cytosolic phosphoprotein, parafusin, that plays a role in regulated exocytosis in the unicellular eukaryote Paramecium (1, 2) and that is evolutionarily conserved (3). Parafusih has been shown to be phosphorylated via a Ca2+-dependent protein kinase (4). Surprisingly, parafusin is also a phosphoglycoprotein in which a short chain ofmannose residues is 0-linked to serine. This chain is phosphoglucosylated by a glucose-1-phosphate phosphotransferase that uses UDP glucose (5). We have recently demonstrated that dephosphoglucosylation is catalyzed by a Ca2+-activated phosphodiesterase. Cells in which parafusin is normal but that are unable to release the content of their dense core secretory vesicles upon stimulation show inactive phosphodiesterase, suggesting that dephosphoglucosylation is a critical event in the pathway to exocytosis (4).Tryptic digests of parafusin purified as described earlier (6)