A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of wholecell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena.The ongoing accumulation of vast collections of DNA sequence data has catalyzed the development of novel approaches for profiling the expression of genes at the mRNA level. These methods, while extraordinarily powerful, do not provide direct information on changes, either in the levels of proteins or their states of modification. The development of analogous high throughput methods for directly monitoring protein levels, while increasingly desirable for biological investigations in the postgenome era (1-3), presents a formidable analytical challenge. Although recent advances in the use of mass spectrometry (MS) in conjunction with protein͞DNA-sequence database search-algorithms allow for the identification of proteins with unprecedented speed (4-7), it remains difficult to obtain accurate quantitative information concerning the levels of the identified proteins and the levels of site-specific modifications within individual protein molecules. In the absence of appropriate antibodies, quantitation is usually achieved by autoradiography after metabolic radiolabeling, fluorography, or the use of protein stains. These procedures depend on complete separation of the proteins of interest by techniques such as high-resolution two-dimensional electrophoresis (8, 9). There remains a pressing need for easier, more reliable means to rapidly profile protein levels. Here we describe a general method for accurately comparing levels of individual proteins present in cell pools that differ in some respect from one another (e.g., the presence of a mutated gene) and for accurately determining changes in the levels of modifications (e.g., phosphorylation) at specific sites on the individual proteins. The procedure can be applied to mixtures of proteins, obviating the need for complete separation. MATERIALS AND METHODSMatrix-Assisted Laser Desorption͞Ionization Mass Spectrometric (MALDI-MS) Tryptic Maps. MALDI-MS (10) tryptic maps of protein gel-bands were obtained as follows.Individual protein bands were excised, destained, washed, and digested with modified trypsin (Boehringer Mannheim), and the resulting peptides were extracted with acetonitrile. After vacuum drying, each sample was redissolved in 5 l ...

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.The Start transition in the Saccharomyces cerevisiae cell cycle requires the activity of one of three cyclin homologs encoded by the CLN1, CLN2, and CLN3 gene family, complexed with the cyclin-dependent kinase encoded by the CDC28 gene. Although the CLN genes are functionally redundant for cell cycle Start (39), the CLN3 gene differs sharply from the CLN1-CLN2 gene pair in structure and regulation (see reference 7 for a review). Searches for mutations resulting in lethality in strains deficient in CLN1 and CLN2 have yielded mutations in a wide range of genes, with widely varying lethal phenotypes. The general conclusion from this result has been that Cln1 and Cln2 are potent at execution of various cell biological processes associated with Start (e.g., cell polarization [4,12] and septin ring formation [4,18]), while Cln3 is relatively weak at activating these processes. Mutations in one gene (BCK2) are lethal in the absence of CLN3 but not in the absence of CLN1 and CLN2. The latter defect is significantly but not completely rescued by placing CLN2 under the control of heterologous promoters (14, 20), as Bck2 is required for efficient transcriptional activation of CLN1 and CLN2 in the absence of CLN3. This result is consistent with other data which suggest that Cln3 is the main or only physiological activator of CLN1 and CLN2 transcription (15,43,44). These r...

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.Cell cycle transitions in eukaryotic cells require the activity of one or more cyclin-dependent protein kinases (CDKs). These kinases are regulated by a family of molecules known as cyclins that share a region of sequence homology termed the cyclin box. Recently, it was shown that the cyclin boxes of human cyclin A, bovine cyclin A, and human cyclin H form five alpha helices that fold into a core domain termed the cyclin fold (3,15,16). Each of these cyclins also contains a second cyclin fold C terminal to that formed by residues of the cyclin box, although the primary sequence of the second cyclin fold contains little homology to either the first cyclin fold or similar regions in other members of the cyclin family. The cyclin box is a major component of the cyclin A-CDK2 interface. Binding of the cyclin molecule results in conformational changes in CDK2, resulting in activation of the kinase (15).Transition through the start of the Saccharomyces cerevisiae cell cycle requires the action of one of three G 1 cyclins, encoded by the CLN1, CLN2, and CLN3 genes, that complex with the CDK encoded by the CDC28 gene. Although the three CLN genes are functionally redundant for cell cycle initiation, CLN3 differs from CLN1 and CLN2 in structure and regulation (reviewed in reference 6). A number of recent studies have determined that the cellular roles of Cln2 and Cln3 also vary, as Cln3 is more efficient at stimulation of transcription at Swi4/Swi6 cell cycle box (SCB) sites and Cln2 is more important for initiation of bud formation and other start-specific cell cycle events (9,20,30). These differences are apparent in a number of genetic assays (20). For example, when expressed from th...

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