Cln Proteins Research Articles

The conserved cellular roles of CLN proteins: Novel insights from Dictyostelium discoideum

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children. The etiology of Batten disease is linked to mutations in 13 genes that encode distinct CLN proteins, whose functions have yet to be fully elucidated. The social amoeba Dictyostelium discoideum has been adopted as an efficient and powerful model system for studying the diverse cellular roles of CLN proteins. The genome of D. discoideum encodes several homologs of human CLN proteins, and a growing body of literature supports the conserved roles and networking of CLN proteins in D. discoideum and humans. In humans, CLN proteins have diverse cellular roles related to autophagy, signal transduction, lipid homeostasis, lysosomal ion homeostasis, and intracellular trafficking. Recent work also indicates that CLN proteins play an important role in protein secretion. Remarkably, many of these findings have found parallels in studies with D. discoideum. Accordingly, this review will highlight the translatable value of novel work with D. discoideum in the field of NCL research and propose further avenues of research using this biomedical model organism for studying the NCLs.

First person – Seda Yasa

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Seda Yasa is first author on ‘CLN3 regulates endosomal function by modulating Rab7A–effector interactions’, published in JCS. Seda is a PhD student in the lab of Stephane Lefrancois at the INRS, Québec, Canada, investigating the role of CLN proteins in intracellular trafficking pathways to develop novel therapeutic strategies for lysosomal storage disorders.

Neuronal ceroid lipofuscinosis genes, CLN2, CLN3 and CLN5 are spatially and temporally co-expressed in a developing mouse brain

Neuronal ceroid lipofuscinosis (NCL) diseases consist of a group of genetically inherited neurodegenerative disorders that share common symptoms such as seizures, psychomotor retardation, blindness, and premature death. Although gene defects behind the NCL diseases are well characterized, very little is known how these defects affect normal development of the brain and cause the pathology of the disease. To obtain understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN2, CLN3 and CLN5 genes was investigated in developing mouse brain. The relationship between the expression pattern and the developmental stage of the brain showed that these genes are co-expressed spatially and temporally during brain development. Throughout the development strong expression of the three mRNAs was detected in germinal epithelium and in ventricle regions, hippocampus and cerebellum, all representing regions that are known to be associated with the formation of new neurons. More specifically, RT-PCR studies on developing mouse cortices revealed that the CLN genes were temporally co-expressed in the neural progenitor cells together with known stem cell markers. This suggested that CLN2, CLN3 and CLN5 genes may play an important role in early embryonal neurogenesis.

The ubiquitin ligase SCFGrr1 is necessary for pheromone sensitivity in Saccharomyces cerevisiae

The presence of the appropriate pheromone induces alpha and a cells of the yeast Saccharomyces cerevisiae to activate both changes in transcriptional expression and cell polarity that eventually lead to the mating of alpha and a cells to form a/alpha diploid cells. A third response after exposure to mating pheromone is a transient cell cycle arrest, allowing synchronization of the two cell types in G1 prior to cell fusion. At least in part, this cell cycle arrest requires the inactivation of Cln-kinase activity through transcriptional inactivation of the CLN1 and CLN2 genes, degradation of the Cln proteins and direct inhibition of Cln-kinase complexes. Here we report that GRR1, which encodes a substrate recognition subunit of SCF complexes, is critical for pheromone sensitivity and likely for this arrest. Loss of SCF(Grr1) function by deletion of the GRR1 gene causes pheromone resistance. However, deletion of CLN1 and CLN2 restores pheromone sensitivity to grr1Delta cells. Thus, rapid loss of Cln-kinase activity during mating may require coordinated inactivation of the Cln-kinase complexes, inactivation of CLN transcription and SCF(Grr1)-dependent Cln degradation.

Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities.

The three budding yeast CLN genes appear to be functionally redundant for cell cycle Start: any single CLN gene is sufficient to promote Start, while the cln1 cln2 cln3 triple mutant is Start defective and inviable. Both quantitative and apparently qualitative differences between CLN genes have been reported, but available data do not in general allow distinction between qualitative functional differences as opposed to simply quantitative differences in expression or function. To determine if there are intrinsic qualitative differences between Cln proteins, we compared CLN2, CLN3, and crippled (but still partially active) CLN2 genes in a range of assays that differentiate genetically between CLN2 and CLN3. The results suggest that different potencies of Cln2, Cln3, and Cln2 mutants in functional assays cannot be accounted for by a simple quantitative model for their action, since Cln3 is at least as active as Cln2 and much more active than the Cln2 mutants in driving Swi4/Swi6 cell cycle box (SCB)-regulated transcription and cell cycle initiation in cln1 cln2 cln3 bck2 strains, but Cln3 has little or no activity in other assays in which Cln2 and the Cln2 mutants function. Differences in Cln protein abundance are unlikely to account for these results. Cln3-associated kinase is therefore likely to have an intrinsic in vivo substrate specificity distinct from that of Cln2-associated kinase, despite their functional redundancy. Consistent with the idea that Cln3 may be the primary transcriptional activator of CLN1, CLN2, and other genes, the activation of CLN2 transcription was found to be sensitive to the gene dosage of CLN3 but not to the gene dosage of CLN2.

Cdc53 Targets Phosphorylated G1 Cyclins for Degradation by the Ubiquitin Proteolytic Pathway

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2–Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin–protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin–proteasome pathway.

G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast.

Entry into a new cell cycle is triggered by environmental signals at a point called Start in G1 phase. A key regulator of this transition step in yeast is the CDC28 kinase together with its short-lived regulatory subunits called G1-cyclins or CLN proteins. To identify genes involved in G1-cyclin degradation, we employed a genetic screen by selecting for stable CLN1-beta-galactosidase fusion proteins. Surprisingly, one group of mutants was found to be allelic to GRR1, a gene previously described to be involved in glucose uptake, glucose repression, and divalent cation transport. In grr1 mutants, both CLN1 and CLN2 cyclins are significantly stabilized. A suppressor analysis indicated that G1-cyclin stabilization in grr1 was not a consequence of the nutrient uptake defect. This suggests that the GRR1 gene product is part of a common regulatory pathway linking two functions important for cell growth, nutrient uptake, and G1 cyclin-controlled cell division.

Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast.

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.

Transcription factors important for starting the cell cycle in yeast

Unlike early embryonic cleavage divisions in certain animals, cell-cycle progression in yeast and probably also in all metazoan somatic cells requires the periodic transcriptional activation of certain key genes. Thus far, the only clear examples are genes that encode a class of unstable 'cyclin' proteins, which bind and activate the cdc2/Cdc28 protein kinase: the G1-specific cyclins encoded by CLN1 and CLN2, a B-type cyclin implicated in DNA replication encoded by CLB5; and four B-type cyclins involved in mitosis encoded by CLB1, 2, 3, 4. CLN1, CLN2, and CLB5 are transcribed in late G1, as cells undergo Start. A transcription factor composed of Swi4 and Swi6 proteins (called SBF) activates CLN1 and CLN2 transcription via a positive feedback loop in which Cln proteins activate their own transcription. A different but related transcription factor called MBF seems responsible for the late G1-specific transcription of most DNA replication genes including CLB5. We have purified MBF and shown that it contains Swi6 and a 110-120 kDa protein distinct from Swi4 (p120) that contacts DNA. Thus, we propose that SBF and MBF share a common regulatory subunit (Swi6) but recognize their promoter elements via distinct DNA binding subunits.

The yeast Cln3 protein is an unstable activator of Cdc28.

The Cln3 cyclin homolog of Saccharomyces cerevisiae functions to promote cell cycle START for only a short time following its synthesis. Cln3 protein is highly unstable and is stabilized by C-terminal truncation. Cln3 binds to Cdc28, a protein kinase catalytic subunit essential for cell cycle START, and Cln3 instability requires Cdc28 activity. The long functional lifetime and the hyperactivity of C-terminally truncated Cln3 (Cln3-2) relative to those of full-length Cln3 are affected by mutations in CDC28: the functional lifetime of Cln3-2 is drastically reduced by the cdc28-13 mutation at the permissive temperature, and the cdc28-4 mutation at the permissive temperature completely blocks the function of Cln3-2 while only partially reducing the function of full-length Cln3. Thus, sequences in the C-terminal third of Cln3 might help stabilize functional Cdc28-Cln3 association, as well as decreasing the lifetime of the Cln3 protein. These and other results strongly support the idea that Cln proteins function to activate Cdc28 at START.

The Yeast Cln3 Protein is an Unstable Activator of Cdc28

The Cln3 cyclin homolog of Saccharomyces cerevisiae functions to promote cell cycle START for only a short time following its synthesis. Cln3 protein is highly unstable and is stabilized by C-terminal truncation. Cln3 binds to Cdc28, a protein kinase catalytic subunit essential for cell cycle START, and Cln3 instability requires Cdc28 activity. The long functional lifetime and the hyperactivity of C-terminally truncated Cln3 (Cln3-2) relative to those of full-length Cln3 are affected by mutations in CDC28: the functional lifetime of Cln3-2 is drastically reduced by the cdc28-13 mutation at the permissive temperature, and the cdc28-4 mutation at the permissive temperature completely blocks the function of Cln3-2 while only partially reducing the function of full-length Cln3. Thus, sequences in the C-terminal third of Cln3 might help stabilize functional Cdc28-Cln3 association, as well as decreasing the lifetime of the Cln3 protein. These and other results strongly support the idea that Cln proteins function to activate Cdc28 at START.

Positive feedback in the activation of Gl cyclins in yeast

Yeast cells become committed to the mitotic cell cycle at a stage during G1 called Start. To enter Start, cells must grow to a critical size. They also require the CDC28 protein kinase and at least one of three G1-specific cyclins encoded by CLN1, 2, and 3. It is thought that Start is triggered by the accumulation of G1 cyclins that bind to the CDC28 kinase and activate it. So what determines the accumulation of G1 cyclins? For CLN1 and CLN2, transcriptional activation could be involved because their RNAs appear transiently during the cell cycle as cells undergo Start. Here we report that the appearance of CLN1 and CLN2 RNAs depends on an active CDC28 kinase and is stimulated by CLN3 activity. We propose that CDC28 kinase activity due to CLN1 and CLN2 proteins arises through a positive feedback loop which allows CLN proteins to promote their own synthesis.

G1-specific cyclins of S. cerevisiae: Cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase

The S. cerevisiae CLN genes encode cyclin homologs essential for progression from G1 to S phase. The CLN2 gene encodes a 62 kd polypeptide that accumulates periodically, peaking during G1 and decreasing rapidly thereafter, and is rapidly lost following exposure of cells to mating pheromone. Cln2 abundance can be explained by the G1-specific accumulation of the CLN2 transcript coupled with instability of the Cln2 protein. The abundance of the CLN1 and CLN2 transcripts increases greater than 5-fold during the G1 interval, decreasing dramatically as cells enter S phase. Both transcripts decrease in cells responding to mating pheromone. Finally, we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex. This physical interaction is consistent with the genetic interaction between the CLN genes and CDC28 and suggests that Cln proteins are an essential component of the active protein kinase complex required for the G1 to S transition.

An essential G1 function for cyclin-like proteins in yeast

Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1 WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1 WHI1 has been renamed CLN3.