Regulation during sulfur deprivation in Chlamydomonas:

I have been intrigued by sulfur deprivation for a long time. Many people have come to the conclusion that while phosphorus and nitrogen often limit the growth of organisms in the environment, sulfur is almost never considered to be limiting. While this is often the case, it should be kept in mind that while a number of environments may have significant sulfur levels, much of this sulfur is not readily available to organisms because it is insoluble or tightly bound to soil particles. Furthermore, as we have explored this area more, we found that there is an enormous diversity of acclimation processes associated with sulfur deprivation in Chlamydomonas and in vascular plants; everything from making new transporters to restructuring the cell wall (which is initially sulfur-rich) and the photosynthetic apparatus. We have used both genetics and biochemistry to explore this regulation and have exploited the finding that when Chlamydomonas is starved for sulfur it makes an extracellular arylsulfatase (ARS) that can be colorimetrically assayed (it cleaves X-SO42-, which, analogous to X-gal, generates a blue precipitate). Below, I have summarized some of our work in this area.
A. Mutant screen: To date, 30,000 paromomycin-resistant Chlamydomonas transformants were screened for aberrant ARS activity (Pollock et al., 2005), which reflects the ability of the cells to acclimate to sulfur deficiency. The gene with the lesion will be tagged (much of the time) with the gene encoding paromomycin resistance, which makes it easy to identify the mutated gene. Many mutants were isolated and characterized (Davies et al., 1996; Pollock et al., 2005; Gonzalez-Ballester et al., 2008; Gonzalez-Ballester et al, 2009; Gonzalez-Ballester, 2010)
B. Analysis of mutant strains: We have gained a lot of experience in analyzing changes in growth, pigmentation and metabolic processes such as photosynthesis, during sulfur starvation, and have also used genetic, molecular and genomic approaches to identify genes encoding regulatory elements that govern sulfur-deprivation responses (Davies et al., 1996; Davies et al., 1999; Eberhard et al., 2006; Gonzalez-Ballester et al., 2009; Pollock et al., 2005; Schwarz et al., 1998; Takahashi et al., 2001; van Waasbergen et al., 2002; Zhang et al., 2004). Recently we have performed RNA-seq with wild-type and strains defective for acclimation to sulfur deprivation. This has provided us with many new insights about changes in metabolism that occur during sulfur deprivation, how sulfur is recycled in the cells and how the cell architecture is reorganized in ways that promote fitness when there is little sulfur in the environment (Gonzalez-Ballester et al., 2010). New results concerning the mutants are summarized below:
1.    The ars11 and ars44 mutants define a serine threonine kinase, SNRK2.1, that functions in the pathway critical for the control of sulfur-deprivation-regulated responses. This mutant is almost completely nonresponsive to sulfur deprivation and is epistatic to two previously isolated mutants, sac1 and sac3 (it is the most stringent non-responder that we have identified). SAC1 is a hierarchical regulator that might function as the sensor for the system while SAC3 is a serine-threonine kinase that functions to repress sulfur-starvation responsive genes under nutrient-replete conditions. These results demonstrate the importance of a phosphorylation cascade(s) in the cellular responses to S deprivation.
2.    Other strains of particular interest are ars401, ars63/ars107 and ars75. The ars401 strain has a lesion in a gene encoding a putative guanylyl cyclase. A cosmid clone containing this guanylyl cyclase gene rescues the mutant phenotype, although there were a few other genes on the complementing fragment (Pollock, unpublished). Interestingly, there are nearly 60 putative guanylyl/adenylyl cyclases encoded on the Chlamydomonas genome. It will be fascinating to elucidate the specificity of expression of these cyclase genes, the biological processes to which they are linked, and the ways in which they integrate into the regulatory circuitry of the cell. The ars63 and ars107 mutants are in a gene encoding a putative SEC24 protein. SEC24 is part of the SEC24/SEC23 complex that selects cargo for capture by membrane vesicles and subsequent export of the cargo from the cell. While both mutants with insertions in SEC24 appear normal under S-sufficient conditions, they do not produce active ARS, suggesting that the SEC24 gene is either required for the maturation and/or secretion of the ARS (and perhaps other extra-cellular proteins). The ars75 mutant is interrupted in a gene encoding a regulatory factor with an ankyrin repeat; the encoded protein may be the transcriptional regulator that directly binds the DNA of the responsive genes. Many of the other mutated genes are also very interesting (e.g. SCF-mediated ubiquitination, ars122; Pho81p-like regulatory proteins, ars5; APPLE domain protein, ars76), although they are all represented by a single mutant allele and it remains to be tested whether or not the lesions in these genes are responsible for the mutant phenotype. Finally, approximately half of the genes identified in this screen encode proteins of unknown function, which we feel should begin to receive significant attention.
C. Sulfate transporters: We have identified six genes encoding putative SO42- transporters in Chlamydomonas and showed that a number of them are up-regulated during sulfur deprivation conditions; three of these transporters are animal-like (SLT1,2,3) and three are plant-like (SULTR1,2,3). Both SULTR2 and SLT2 migrate as doublets in gels (both are up-regulated during sulfur deprivation), suggesting post-translational modifications. We have monospecific antibodies for SULTR2 and SLT2, are preparing monospecific antibodies to SLT1, SLT3 and SULTR1, and also have family-specific (SLT and SULTR) antibodies. Recently we demonstrated that SULTR2, SLT2 and SLT1 accumulate at the protein level during sulfur deprivation; all three are localized to the plasma membrane. Furthermore, we have been able to generate and inactivation in each of these transporters and make double mutants and the triple mutant in which all three of the transporters are inactivated. In this latter strain the cells show no increase in SO42-transport when they are starved for sulfur, demonstrating that these three transporters are responsible for all of the sulfur deprivation inducible transport observed in wild-type cells (Pootakham et al., 2010).
D. Effect of cycloheximide on gene expression: Recently, we have demonstrated that the levels of transcripts from many of the sulfur-responsive genes are sensitive to the protein synthesis inhibitor cycloheximide (CHX). The transcripts for SULTR2, SLT1, SLT2, SBDP, ARS1, and ECP76 increase when the cells are starved for sulfur. The abundance of all of these transcripts increases from between 100-fold to several 1,000-fold relative to their initial level (after 6-12 h of sulfur-deprivation). Interestingly, while the transcripts for all transporters appear to increase to a greater extent when the cells are treated with CHX (the increase might reflect protection of the transcripts bound to ribosomes), CHX completely blocks accumulation of ARS1, ECP76 and SBDP mRNA. Chloramphenicol (Cm) has essentially no effect on transcript accumulation. These results suggest that there are at least two tiers of transcriptional control associated with sulfur-deprivation responses. Immediately following sulfur deprivation the genes encoding the transporters are activated (their transcripts are detected 30 min to 1 h after the imposition of sulfur deprivation) in a protein synthesis-independent manner, followed by the rapid synthesis of the transporter polypeptides (the proteins are detected within 1 h of removing SO42- from the medium); this represents the first tier of gene activation. This first tier protein synthesis is required for second tier gene activation (represented by genes such as ARS, ECP76 and SBDP). A candidate for the protein(s) required for activation of the second tier (based on the mutant phenotype) has recently been identified; some features of this protein suggest that it may be involved in regulating the transcription of genes.