The Genetic Engineering Proposal Project, an IBI prize-winning module, teaches biology students to devise innovative bioproducts or solutions to environmental or health problems.

ABSTRACT Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7 , CUE1 , or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Δ cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7 , CUE1 , or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.

CBE—Life Sciences EducationVol. 5, No. 4 FeaturesFree AccessWalking the WalkRobin L. WrightRobin L. WrightDepartment of Genetics, Cell Biology and Development, University of Minnesota, St. Paul, MN 55108Search for more papers by this authorPublished Online:17 Oct 2017https://doi.org/10.1187/cbe.06-05-0163AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Of the dozens of books about teaching and learning that I have read over the past decade, none has reverberated in my thoughts like Learner-Centered Teaching. This book confronted me with the great distance between my personal definition and practice of “learner-centered teaching” and the potential of a deeper, more authentic learner-centered approach. It confronted me with the lapses between my “talk” and my “walk” when it comes to attitudes toward students, course design, expectations, power, and many other aspects of teaching. It helped me think more creatively about helping students with diverse learning styles and needs to thrive in my classes. What makes this book so valuable and thought provoking?Weimer begins her book with a discussion of her redesign of a communications course to be more learner centered. In this new course, students were provided with a menu of assessment options, so that specific decisions about how they would be evaluated were determined by the student herself or himself. Based on my own teaching experience, the initial response of Weimer 's students was predictable: They were stunned and uncomfortable, looking for the “catch.” They could not initially believe that they did not have to take the exams, but could earn their points in other ways. The ultimate response of her students was less predictable but not surprising in hindsight: Students became more engaged with the material and worked harder than they did in Weimer's previous teacher-centered course. This anecdote, told in a pleasant conversational style, leaves the reader with a strong desire to design and teach similar courses, courses in which students find the content interesting and relevant, are animated in class discussions, and learn more effectively.The course itself is also very interesting as a model for creative learning activities. For example, students in the class could earn points by “class participation.” Each class participant was paired with another participant, with each member acting as a participation “coach” for the other. The class as a whole defined what class participation meant, and the participants earned their points by writing three papers: a paper outlining their participation goals in the context of the class definitions, a feedback paper to their partner about his or her class participation, and a self-assessment of his or her own participation. Such an assignment/assessment plan involving student coaches was delightfully new to me and made me consider other ways in which my students might participate in feedback to one another.In the next five chapters, Weimer presents five changes that she believes are necessary to create an authentic learner-centered teaching practice: 1) shifting the balance of power more toward the student; 2) using (rather than covering) content as the means to achieve higher-order learning goals; 3) changing the teacher's role from that of telling/doing to that of designing/modeling; 4) helping students accept the responsibility for learning; and 5) adapting the purpose and processes of evaluation to promote learning. Maintaining the highly readable, conversational writing style that draws you in at the beginning, Weimer elaborates on each of these changes in a separate chapter. Each chapter ends with a very effective summary paragraph that reinforces the key messages of the chapter. Because the chapters are short and focused and the key messages are restated at the end, the reader falls almost automatically into a pattern of intense reading, followed by reflection. In my case, the book was read over a couple of weeks of commutes in the “Campus Connector” between Minneapolis and St. Paul. It wasn't that the book could not be read at one sitting. Instead, after reading each chapter, I found myself needing to internalize the information and to envision the changes that I might make to my own teaching. How should I structure my new seminar to give students more decision-making power? How could I help students assume greater responsibility for their learning? How could I design assessments that help students actually master the course content? How would students respond to these changes?Although the first part of the book is essentially a detailed explanation of learner-centered teaching, the second part deals with how to implement the necessary changes to achieve that goal. Surprisingly, the implementation part of the book was less valuable than the definition part. For example, as I'm sure the readers of CBE—Life Sciences Education recognize, changes such as those recommended above are likely to cause resistance from every quarter, including from both students and faculty colleagues. Weimer discusses where the resistance arises. For example, students resist learner-centered approaches because these approaches require more work, are more threatening than traditional teacher-centered approaches, force students to take responsibility for their learning, and may be difficult for some students. Weimer's answer to this resistance is communication with students. Thus, although the analysis of the source of resistance was valuable, the solutions appeared superficial. Nevertheless, this portion of the book forces faculty who are considering moving toward learning-centered teaching to understand some of the challenges that face them.After reading this book and pondering on its message for several months, I returned to it recently and revisited key points. The ideas and conversational style still resonate with great power: the function of content as a vehicle for skill building; the teacher as midwife, trail guide, or coach; the roles of self-assessment, formative assessment, and grades. Powerful ideas. Difficult ideas. Revolutionary ideas.I reconsidered my own attitudes toward teaching: I envision my courses to epitomize the “guide on the side” style of teaching. I pat myself on the back as I include bits of active learning exercises or “clicker questions” at the midpoint of the 30 PowerPoint slides that I need to cover in that day's lecture. I'm proud of the tight organization of our courses that provide students with clear expectations from the time they enter the course.Although these changes are authentic improvements since my first dismal teaching experiences (I'm still profoundly sorry for those first students!), Learner-Centered Teaching puts these efforts into an intense new light. It reveals that I still have a long way to go! Few books on education have had the impact of Learner-Centered Teaching on my thinking about how to be a better teacher. And, although I'm not yet truly walking the walk, I think I'm at least up on all fours and moving ahead.FiguresReferencesRelatedDetailsCited ByLearner-centered Approach in Teaching Foreign Language: Psychological and Pedagogical ConditionsProcedia - Social and Behavioral Sciences, Vol. 206Using Critical-Thinking Skills to Substantiate or Challenge Controversial Claims Endorsing a Myriad of Weight-Loss ProductsThe American Biology Teacher, Vol. 72, No. 9 Vol. 5, No. 4 December 01, 2006297-360 Metrics Downloads & Citations Downloads: 73Citations: 2 History Information© 2006 by The American Society for Cell BiologyPDF download

Cell Biology EducationVol. 4, No. 4 FEATURESFree AccessA 15-Year Study of 63 Teachers at 24 Institutions Reveals:“ What the Best College Teachers Do”Robin L. Wright, Aaron Charlson, and Carrie F. OlsonRobin L. WrightSearch for more papers by this author, Aaron CharlsonSearch for more papers by this author, and Carrie F. OlsonSearch for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1187/cbe.05-08-0112AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail A PROFESSOR'S VIEWIn this brief but inspiring book, Ken Bain, director of the Center for Teaching Excellence at New York University, reports results of a 15-year analysis of the scholarship and practices of “the best college teachers in the United States.” Although Bain has been interested in understanding the practices of best teachers for more than 40 years, this study was catalyzed by the suicide of Tom Philpott, a gifted teacher whose“ library of teaching talents and practices burned to the ground when he died.” Bain's motive was to capture and distill the collective wisdom and experience of exceptional college teachers so that their accumulated wisdom did not evaporate when they were lost to us.The first chapter of the book provides an executive summary of the study and its conclusions. Bain defines the best teachers as those who have a“ sustained, substantial, and positive influence on how [their] students think, act, and feel.” He briefly describes how he identified a cohort of 63 (mostly) unnamed college teachers who met these criteria, based largely on analysis of student evaluations for evidence of “deep learning.” The practices of teachers who made the “best teacher” list, as well as their students, were studied through interviews, statements of their teaching philosophy, observations, analysis of course materials and student work, and comments from colleagues.In spite of an overall weakness in describing details of how teachers were selected and studied, the conclusions ring true: 1) The best college professors know their subjects extremely well and understand human learning. 2) They prepare to teach with the same rigor and dedication that they bring to their research endeavors. 3) They have high expectations of their students. 4) They create a “natural critical learning environment” in which students confront important problems in ways that force them to rethink assumptions and “examine their mental models of reality.” 5) They have deep respect for their students, including an assumption that their students are both able and eager to learn. 6) They assess their own teaching effectiveness and make changes to their approaches based on these data.The remaining chapters of the book elaborate on these conclusions, using many examples from actual classrooms. These examples and anecdotes make the book a great pleasure to read and provide a great deal of its value. In fact, it is one of those books from which I have found new insights and inspiration on my second and third readings. For example, Chapter 2 summarizes our current understanding of human learning, including examples of creative ways in which the best college teachers apply this understanding to their classes. Bain explains the importance of motivating long-term learning through intrinsic rather than extrinsic rewards (usually grades). He then describes ways in which the best college teachers apply this motivating principle in their classes. Among other actions, they avoid assigning grades based on averages of performance throughout the course, provide many and varied opportunities for students to demonstrate mastery, and focus on the subject itself rather than logistical nuts and bolts on the first day of class. Reading this section reinforced my growing discomfort with my own reliance on summative assessment of student learning. Why shouldn't a student who develops a sophisticated understanding of mitosis and meiosis by the end of the course receive the same grade on that subject as a student who gains that understanding earlier in the course? Based on this insight, I now count the final exam grade in lieu of exam averages if it provides students with a higher grade. (Of course, this practice brings up the myriad of problems associated with high-stakes exams and the eternal but unfounded optimism of students that they can always simply ace the next exam. I'm still grappling with these issues.)The major inadequacy of the book is its minimal description of the teacher selection criteria and methods of analysis. Although more detail is provided in an appendix, the descriptions appear superficial, at least to my sensibilities. In addition, there appear to be no peer-reviewed articles generated by this study to which one could turn for detailed experimental information. In fact, few references of any kind are provided to the reader, and these are confined to a cumbersome “notes” section. The lack of experimental detail is a flaw of the book that creates a deep wound of missed opportunity. For example, on its own, providing a mechanism for using teaching evaluations for more than a popularity poll would have been of great value. Without these details, the collective wisdom of this clearly talented and insightful student of great teachers and great teaching runs the risk of being lost in much the same way as the teaching wisdom of Tom Philpott.Still, in spite of these limitations, this book provided me with useful, practical insights about how I can improve my teaching, even if I never make the best college teacher list. More importantly, it continues to inspire me (now on my fourth reading!) that I can develop more effective ways to support my students' learning, even within the constraints of the limited time I have available to devote to my teaching. This ongoing value has earned What the Best College Teachers Do a special place on my bookshelf.THE VIEWS OF TWO UNDERGRADUATES1When we were invited to review What the Best College Teachers Do, we wondered, “How can we, as students, give any insight into what is involved in good teaching?” Surprisingly, we found that this book helped us not only to think more critically about excellent teaching but also to reflect on our own learning. We began to see that our impressions of previous instructors, both good and bad, reflected the criteria the book used to define good teaching. We almost felt inspired to begin writing letters to past teachers either thanking them for taking these factors into account when preparing to enter the classroom or pleading with them to read this book and think about how their own skills could improve by applying these qualities to their lesson plans.1Aaron T. Charlson and Carrie F. Olson are undergraduates at the University of Minnesota. Mr. Charlson is a junior double-majoring in Genetics and Microbiology; Ms. Olson is a junior double-majoring in Genetics and Ecology. Both have been engaged in undergraduate research for several years and share an interest in the undergraduate educational experience, as evidenced by their serving as peer mentors for incoming freshmen. Collectively, they have accumulated more than 8,000 hours in college classes and been taught by approximately 40 different professors. They would like to thank the teachers who have inspired and challenged them; these teachers taught them a great deal about the subject but, more importantly, helped them learn even more about themselves.One result of the study that particularly struck us was how Bain selected the best teachers and, consequently, how he defined the best teaching practices. It was a relief to find that the definition of good teaching doesn't just involve how well a particular group of students, usually the upper crust, does on a test. Instead, good teaching involves bringing about inner changes in the student, such as helping students develop a new appreciation of the topic and a desire to pursue it further. It seems too often, at least from our student perspective, that the professor is simply going through the motions to cover the required material without even trying to inspire students to explore beyond the boundaries defined by the expected tests. Our best teachers have had the attitude, emphasized in this book, that it is not sufficient that a student remembers some facts or formulas but that the student learns how to use them. Our experience agrees with the book's definition of what great teaching should encompass: the passion of the professor being passed on to the student.This definition of good teaching (and learning) runs counter to the emphasis on memorizing facts that we encounter in many of our classes. From our own experience, we know that what we “learned” in such content-focused classes is quickly forgotten after the final examination, creating a “bulimic” learning cycle. The possibility that so much of our hard work in college can be so easily lost is deeply troubling. As described in the book, our best teachers have helped us avoid the learn-purge cycle by taking us beyond the facts and leading us into an understanding of the topic on a deeper, more intuitive level. As the book describes, they have helped us think deeply by asking questions that challenge our preconceived ideas about the subject and point out the flaws or holes in our current understanding. They have taught us in the context of complex or controversial problems. They have torn down everything we thought we knew and helped us replace it with more refined ideas of the world.We found it very interesting that, as we read this book and thought about best teaching strategies and approaches in general, we also thought about our own learning in ways we hadn't done before. For example, as we evaluated our own best and worst teachers using the principles defined in this book, we began to look for examples of teachers who had had a long-lasting impact on our thinking. Surprisingly, one of these teachers was initially on our“ worst teacher” list. Thus, reading about teaching helped us to think more kindly about our professors as well as to examine our own learning in new ways.The ability of this book to help us reflect on our own learning was particularly valuable. In fact, although we would recommend this book to our faculty who want to improve their teaching, we also would recommend the parts that describe human learning to students. For example, the idea of constructing knowledge rather than receiving it is a message that is important for students and teachers alike. Although teachers play a crucial role in helping their students understand the applications and implications of the information presented, the student must be a willing participant. After all, that's where the learning actually takes place — in the student's brain! Thus, knowing more about how learning happens could help students assess what type of learning strategy they are using and hone their skills to achieve greater learning, no matter what teaching situation they happen to be in.FiguresReferencesRelatedDetails Vol. 4, No. 4 December 01, 2005261-343 Metrics Downloads & Citations Downloads: 89 History Information© 2005 by The American Society for Cell BiologyPDF download

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

To help students develop successful strategies for learning how to learn and communicate complex information in cell biology, we developed a quarter-long cell biology class based on team projects. Each team researches a particular human disease and presents information about the cellular structure or process affected by the disease, the cellular and molecular biology of the disease, and recent research focused on understanding the cellular mechanisms of the disease process. To support effective teamwork and to help students develop collaboration skills useful for their future careers, we provide training in working in small groups. A final poster presentation, held in a public forum, summarizes what students have learned throughout the quarter. Although student satisfaction with the course is similar to that of standard lecture-based classes, a project-based class offers unique benefits to both the student and the instructor.

Increased expression of certain ER membrane proteins leads to biogenesis of novel ER membrane arrays. These structures provide models in which to explore the mechanisms by which cells control the size and organization of organelles in response to changing physiological demands. In yeast, elevated levels of HMG-CoA reductase induce ER arrays known as karmellae. Cox and co-workers (1997) discovered that karmellae assembly is toxic to ire1 mutants. These mutants are unable to initiate the unfolded protein response, which enables cells to adjust levels of ER chaperones in response to stresses. We sought to determine whether the karmellae-dependent death of ire1 mutants was due to karmellae assembly or to increased levels of HMG-CoA reductase activity. Unexpectedly, we found that ire1 cells could assemble normal levels of karmellae that were structurally identical to those of wild-type cells. In addition, karmellae assembly did not itself induce the unfolded protein response. Certain ire1 strains produced significant numbers of transformants that were unable to utilize galactose as sole carbon source. These results suggest that the karmellae-dependent death of certain ire1 strains may simply reflect their inability to grow on galactose.

In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.

In response to elevated levels of HMG-CoA reductase, an integral endoplasmic reticulum (ER) membrane protein, cells assemble novel ER arrays. These membranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover. Yeast express two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces morphologically different ER arrays. Hmg1p induces stacks of paired nuclear-associated membranes called karmellae. In contrast, Hmg2p induces peripheral ER membrane arrays and short nuclear-associated membrane stacks. In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane domain containing eight predicted transmembrane helices. By examining a series of recombinant HMG-CoA reductase proteins, our laboratory previously demonstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper karmellae assembly. Our goal was to examine the primary sequence requirements within Loop G that were critical for proper function of this signal. To this end, we randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at wild-type levels. Out of approximately 4000 strains with Loop G mutations, we isolated 57 that were unable to induce wild-type levels of karmellae assembly. Twenty-nine of these mutants contained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembly. Comparison of these mutations to single point mutations that did not affect karmellae assembly did not reveal obvious patterns of sequence requirements. For example, both conservative and non-conservative changes were present in both groups and changes that altered the total charge of the Loop G region were observed in both groups. Our hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be important for maintaining the proper secondary structure needed for these interactions.

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well‐fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde‐fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold‐labeled secondary antiserum. Microsc. Res. Tech. 51:496–510, 2000. © 2000 Wiley‐Liss, Inc.

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using beta-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed 'feed-forward' regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin.The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Lipophilic fluorescent dyes are used for examining cell structure and investigating the structure and function of specific organelles. One of these dyes, 3, 3′-dihexyloxacarbocyanide iodide [DiOC6(3)] is used to investigate the properties of the endoplasmic reticulum in living and fixed cells. DiOC6(3) diffuses through the plasma membrane and enters cell membranes, but in living cells the dye becomes concentrated within specific organelles. The concentration of DiOC6(3) determines the organelles who appear stained. This selectivity of DiOC6(3) staining reflects accumulation of the positively charged dye molecules in subcellular membranes that have negative membrane potential. The sensitivity of DiOC6(3) accumulations to membrane potential is used to measure plasma membrane potential in mammalian cells. When Chlamydomonas reinhardtii cells are exposed to low concentrations of DiOC6(3), flagella rather than mitochondria are specifically stained. Staining of mitochondria is observed only when the DiOC6(3) concentrations is increased, and even then, the flagella continue to stain. At the highest DiOC6(3) concentrations tested, other cellular membranes including the chloroplast membrane, also became labeled. If the staining characteristics of DiOC6(3)reflect its membrane potential-sensitive partitioning into membranes, flagellar ion pumps are to maintain the higher negative membrane potential across the flagellar membranes than that of the rest of the plasma membrane, or even that of mitochondria. It is conceivable that this membrane potential may have functional roles in controlling flagellar length, autotomy, or aspects of flagellar beating. There is description of medium and culture conditions, preparation and storage of DiOC6(3), staining of Chlamydomonas with DiOC6(3), microscopy, staining patterns and prospects. Long-chain dyes, once incorporated into a membrane, the dye is retained within that membrane. DiOC6(3) staining may be revealing physiologically relevant information concerning flagellar membrane potential. Use of an emission filter that blockslight from 555 to 1.2 nm eliminates chlorophyll autofluorescence, a necessity photography on a standard light microscope.