Different ER-plasma membrane tethers play opposing roles in autophagy of the cortical ER.

The endoplasmic reticulum (ER) undergoes degradation by selective macroautophagy (ER-phagy) in response to starvation or the accumulation of misfolded proteins within its lumen. In yeast, actin assembly at sites of contact between the cortical ER (cER) and endocytic pits acts to displace elements of the ER from their association with the plasma membrane (PM) so they can interact with the autophagosome assembly machinery near the vacuole. A collection of proteins tether the cER to the PM. Of these, Scs2/22 and Ist2 are required for cER-phagy, most likely through their roles in lipid transport, while deletion of the tricalbins, TCB1/2/3, bypasses those requirements. An artificial ER-PM tether blocks cER-phagy in both the wild type (WT) and a strain lacking endogenous tethers, supporting the importance of cER displacement from the PM. Scs2 and Ist2 can be cross-linked to the selective cER-phagy receptor, Atg40. The COPII cargo adaptor subunit, Lst1, associates with Atg40 and is required for cER-phagy. This requirement is also bypassed by deletion of the ER-PM tethers, suggesting a role for Lst1 prior to the displacement of the cER from the PM during cER-phagy. Although pexophagy and mitophagy also require actin assembly, deletion of ER-PM tethers does not bypass those requirements. We propose that within the context of rapamycin-induced cER-phagy, Scs2/22, Ist2, and Lst1 promote the local displacement of an element of the cER from the cortex, while Tcb1/2/3 act in opposition, anchoring the cER to the plasma membrane.

Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.

Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport. To explore this possibility, we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab Ypt51. The Sec2GEF-GFP-CUE construct localized to bright puncta predominantly near sites of polarized growth, and this localization was dependent on the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with various efficiencies. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near-normal efficiency, implying that Golgi-derived secretory vesicles were delivered to polarized sites of cell growth despite the misdirection of Sec4 and its effectors. A low efficiency mechanism for localization of Sec2 to secretory vesicles that is independent of known cues might be responsible. In total, the results suggest that while Rabs may play a critical role in specifying the direction of vesicular transport, cells are remarkably tolerant of Rab misdirection.

Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.

Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport because anterograde vesicles are marked with a different Rab than retrograde vesicles. To explore this proposal, we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab, Ypt51. The Sec2GEF-GFP-CUE construct was found to localize to bright puncta predominantly near sites of polarized growth and this localization was strongly dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. The puncta appeared to consist of clusters of 80 nm vesicles and although the puncta are largely static, FRAP analysis suggests that traffic into and out of these clusters continues. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth, where they tethered and fused with the plasma membrane despite the misdirection of Sec4 and its effectors. In total, the results suggest that while Rabs play a critical role in regulating vesicular transport, cells are remarkably tolerant of Rab misdirection.

Actin assembly at sites of contact between the cortical ER and endocytic pits promotes ER autophagy.

A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex toward the site of autophagosome assembly near the vacuole.

Reconnecting Students and Faculty to Maximize Academic Integrity and Minimize Student Stress in the Virtual Classroom.

The article documents faculty experiences with the shift online due to the pandemic and provides recommendations to science, technology, engineering, and mathematics (STEM) instructors. Over 100 faculty members were surveyed on these topics and contrasted with previously reported student experiences. The online shift changed how faculty administered exams, ran courses, and acted to ensure academic integrity. For example, when exams went online, 73% of faculty reported spending more time preventing cheating. Concerning academic integrity and stress, faculty and students agreed with the exception of a few notable disconnects. Students reported greater workloads in online classes, while faculty maintained that the shift online did not change student workloads. Students perceived more online cheating than faculty. Overall, there seems to be a significant disconnect regarding faculty not realizing how much their actions may encourage or discourage cheating. Few faculty (<15%) indicated that being a tough grader or having test times too short is a motivating factor, but over 55% of students reported that these motivate students to cheat. Conversely, over 60% of students reported respect for their professors discourages them from cheating, while only 37% of faculty indicated the same. Over 70% of faculty and students indicated that fear of getting caught is a deterrent to cheating. Recommendations to reconnect include (i) faculty should use the finding that the number one deterrent of cheating is fear of getting caught; and (ii) faculty should maintain students' respect by being clear or overestimating workload requirements, carefully adjusting time for online exams, and setting clear expectations with uncomplicated exam questions consistent with the material taught.

Double NPY motifs at the N-terminus of the yeast t-SNARE Sso2 synergistically bind Sec3 to promote membrane fusion.

Exocytosis is an active vesicle trafficking process by which eukaryotes secrete materials to the extracellular environment and insert membrane proteins into the plasma membrane. The final step of exocytosis in yeast involves the assembly of two t-SNAREs, Sso1/2 and Sec9, with the v-SNARE, Snc1/2, on secretory vesicles. The rate-limiting step in this process is the formation of a binary complex of the two t-SNAREs. Despite a previous report of acceleration of binary complex assembly by Sec3, it remains unknown how Sso2 is efficiently recruited to the vesicle-docking site marked by Sec3. Here, we report a crystal structure of the pleckstrin homology (PH) domain of Sec3 in complex with a nearly full-length version of Sso2 lacking only its C-terminal transmembrane helix. The structure shows a previously uncharacterized binding site for Sec3 at the N-terminus of Sso2, consisting of two highly conserved triple residue motifs (NPY: Asn-Pro-Tyr). We further reveal that the two NPY motifs bind Sec3 synergistically, which together with the previously reported binding interface constitute dual-site interactions between Sso2 and Sec3 to drive the fusion of secretory vesicles at target sites on the plasma membrane.

Maximizing Academic Integrity While Minimizing Stress in the Virtual Classroom.

The article documents students' experiences with the shift online at the onset of the COVID-19 pandemic and provides informed recommendations to STEM instructors regarding academic integrity and student stress. Over 500 students were surveyed on these topics, including an open-ended question. Students experienced more stress and perceived a greater workload in online courses and therefore preferred in-person courses overall. Personal awareness of cheating during online exams is positively correlated with the proportion of cheating a student perceives. Fear of getting caught is the best cheating deterrent while getting a better grade makes cheating most enticing. Randomization of questions and answer choices is perceived as a highly effective tool to reduce cheating and is reported as the least stress-inducing method. Inability to backtrack and time limits cause students the most stress. Students report that multiple choice questions are the least effective question type to discourage cheating and oral exam questions cause the most stress. Use of camera and lockdown browser or being video- and audio- recorded caused the majority of student stress. Yet, nearly 60% agree that the combination of camera and lockdown browser is an effective deterrent. Recommendations: (i) Be transparent regarding academic dishonesty detection methods and penalties. (ii) Use online invigilating tools. (iii) Synchronize exams and (iv) randomize exam questions. (v) Allow backtracking. (vi) Avoid converting in-person exams to online exams; instead, explore new ways of designing exams for the online environment.

ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits.

Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.

Autophagy of the ER requires actin assembly driven by the interaction of ER with endocytic pits.

Autophagy of the cortical ER in budding yeast was unexpectedly found to require End3, a component of the endocytic machinery that promotes the assembly of actin at endocytic pits on the plasma membrane. The cortical ER transiently interacts with invaginating endocytic pits through a linkage consisting of VAP proteins, oxysterol binding proteins and type I myosins. These proteins are required for actin assembly and for autophagy of the ER. Assembly of actin at these contact sites may direct the movement of ER away from the cortex towards sites of autophagosome assembly.

Testing the Phenotypic Effects of a Rab Chimera that Resolves Exchange Factor Specificity from Effector Specificity.

Rab GTPases play key roles in defining the identity of the various compartments that comprise the secretory and endocytic pathways. Recruitment of a Rab to a specific compartment requires its localized activation by a guanine nucleotide exchange factor (GEF). This in turn results in the recruitment of a distinct set of Rab effectors that directs the recognition of the appropriate target compartment by a carrier vesicle and their subsequent fusion. A chimeric Rab protein, Ypt1-SW1Sec4, was found to separate GEF specificity from effector specificity (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006), but early studies did not observe strong effects of this allele on growth or membrane traffic (Brennwald P, Novick P. Nature 362(6420):560-563, 1993). To resolve this apparent conundrum, yeast strains expressing the chimeric Rab were subjected to a more extensive battery of phenotypic tests. These tests demonstrated that changing the specificity of the GEF interaction does lead to a change in Rab localization and can lead to the ectopic recruitment of an effector, creating trafficking defects that are dependent upon the level of expression (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006). Here we describe the methods used in this analysis. Specifically we describe the following: 1. An assay used to quantify the efficiency of export of a cell wall protein Bgl2, 2. The use of thin section electron microscopy to address the morphology of the secretory machinery, 3. The use of a fluorescently tagged vesicle SNARE protein, GFP-Snc1, to follow plasma membrane recycling and. 4. The use of fluorescently tagged Ypt1 effectors, Cog3-GFP, Uso1-GFP, and Sec7-GFP to follow their recruitment by Ypt1-SW1Sec4.

Triage and Recovery of STEM Laboratory Skills.

The global COVID-19 pandemic left universities with few options but to turn to remote learning. With much effort, STEM courses made this change in modality; however, many laboratory skills, such as measurement and handling equipment, are more difficult to teach in an online learning environment. A cohort of instructors who are part of the NSF RCN-UBE funded Sustainable, Transformative Engagement across a Multi-Institution/Multidisciplinary STEM (STEM)2 Network (a working group of faculty from two community colleges and three 4-year universities) analyzed introductory biology and chemistry courses to identify essential laboratory skills that students will need in advanced courses. Seven essential laboratory proficiencies were derived from reviewing disciplinary guiding documents such as AAAS Vision and Change in Undergraduate Biology Education, the American Society for Microbiology Recommended Curriculum Guidelines for Undergraduate Microbiology Education, and the American Chemical Society Guidelines for Chemistry: data analysis, scientific writing, proper handling and disposal of laboratory materials, discipline-specific techniques, measurement, lab safety and personal protective equipment, and interpersonal and collaborative skills. Our analysis has determined that some of these skills are difficult to develop in a remote or online setting but could be recovered with appropriate interventions. Skill recovery procedures suggested include a skills "boot camp," department or college coordinated club events, and a triage course. The authors recommend that one of these three recovery mechanisms be offered to bridge this skill gap and better prepare STEM students for upper-level science courses and the real world.

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy.

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

GIV/Girdin and Exo70 Collaboratively Regulate the Mammalian Polarized Exocytic Machinery.

Polarized exocytosis is a fundamental process by which membranes and cargo proteins are delivered to the cell surface with precise spatial control. Although the need for the octameric exocyst complex is conserved from yeast to humans, what imparts spatial control is known only in yeast, i.e., a polarity scaffold called Bem1p. We demonstrate here that the mammalian scaffold protein, GIV/Girdin, fulfills the key criteria and functions of its yeast counterpart Bem1p; both bind Exo70 proteins via similar short-linear interaction motifs, and each prefers its evolutionary counterpart. Selective disruption of the GIV⋅Exo-70 interaction derails the delivery of the metalloprotease MT1-MMP to invadosomes and impairs collagen degradation and haptotaxis through basement membrane matrix. GIV's interacting partners reveal other components of polarized exocytosis in mammals. Findings expose how the exocytic functions aid GIV's pro-metastatic functions and how signal integration via GIV may represent an evolutionary advancement of the exocytic process in mammals.

ER-phagy requires Lnp1, a protein that stabilizes rearrangements of the ER network.

The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets that is predominantly associated with the cell cortex in yeast. Upon treatment with rapamycin, the ER undergoes degradation by selective autophagy. This process, termed ER-phagy, requires Atg40, a selective autophagy receptor that localizes to the cortical ER. Here we report that ER-phagy also requires Lnp1, an ER membrane protein that normally resides at the three-way junctions of the ER network, where it serves to stabilize the network as it is continually remodeled. Rapamycin treatment increases the expression of Atg40, driving ER domains marked by Atg40 puncta to associate with Atg11, a scaffold protein needed to form autophagosomes. Although Atg40 largely localizes to the cortical ER, the autophagy machinery resides in the cell interior. The localization of Atg40 to sites of autophagosome formation is blocked in an lnp1Δ mutant or upon treatment of wild-type cells with the actin-depolymerizing drug Latrunculin A. This prevents the association of Atg40 with Atg11 and the packaging of the ER into autophagosomes. We propose that Lnp1 is needed to stabilize the actin-dependent remodeling of the ER that is essential for ER-phagy.

Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane.

The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion. We anchored each subunit with a transmembrane (TM) domain at its N- or C-terminus. Only N-terminally anchored TM-Sec3p and C-terminally anchored Exo70p-TM proved functional. These findings orient the complex with respect to the membrane and establish that Sec3p and Exo70p can function exclusively on the membrane. The functions of TM-Sec3p and Exo70p-TM were largely unaffected by blocks in endocytic recycling, suggesting that they act on the plasma membrane rather than on secretory vesicles. Cytosolic pools of the other exocyst subunits were unaffected in TM-sec3 cells, while they were partially depleted in exo70-TM cells. Blocking actin-dependent delivery of secretory vesicles in act1-3 cells results in loss of Sec3p from the purified complex. Our results are consistent with a model in which Sec3p and Exo70p can function exclusively on the plasma membrane while the other subunits are brought to them on secretory vesicles.

Rewiring a Rab regulatory network reveals a possible inhibitory role for the vesicle tether, Uso1.

Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1Sec4, is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1Sec4 as the sole copy of YPT1 exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1Sec4 We show that even normal expression of Ypt1-SW1Sec4 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1Sec4, the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking.

Regulation of membrane traffic by Rab GEF and GAP cascades.

ASBTRACT Rab GTPases serve as master regulators of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effector proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange factors (GEFs), which catalyze the displacement of GDP and binding of GTP, as well as GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Here I review our studies on the final stages of the yeast secretory pathway that have led us to propose that adjacent Rabs on a pathway are networked to one another through their regulators; specifically we have shown that the Rab, Ypt32, in its GTP-bound form recruits both Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The postulated effect of these counter-current cascades is a programmed series of abrupt Rab transitions that lead to critical changes in the functional identity of the membrane as it flows along the exocytic pathway. Phosphoinositides also play key roles in the temporal and spatial regulation of membrane traffic. The Golgi pool of phosphatidylinositol 4-phosphate (PI(4)P) works in concert with Ypt32 to initially recruit Sec2, yet a subsequent drop in PI(4)P levels directs a regulatory switch in Sec2 function in which it binds to the Sec4 effector Sec15 generating a positive feedback loop. PI(4)P distribution together with Sec2 phosphorylation by a casein kinase determines when and where each regulatory circuit is used.

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p.

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling.

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum.

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.

Evolutionary dynamics of rhomboid proteases in Streptomycetes.

BACKGROUND: Proteolytic enzymes are ubiquitous and active in a myriad of biochemical pathways. One type, the rhomboids are intramembrane serine proteases that release their products extracellularly. These proteases are present in all forms of life and their function is not fully understood, although some evidence suggests they participate in cell signaling. Streptomycetes are prolific soil bacteria with diverse physiological and metabolic properties that respond to signals from other cells and from the environment. In the present study, we investigate the evolutionary dynamics of rhomboids in Streptomycetes, as this can shed light into the possible involvement of rhomboids in the complex lifestyles of these bacteria. RESULTS: Analysis of Streptomyces genomes revealed that they harbor up to five divergent putative rhomboid genes (arbitrarily labeled families A-E), two of which are orthologous to rhomboids previously described in Mycobacteria. Characterization of each of these rhomboid families reveals that each group is distinctive, and has its own evolutionary history. Two of the Streptomyces rhomboid families are highly conserved across all analyzed genomes suggesting they are essential. At least one family has been horizontally transferred, while others have been lost in several genomes. Additionally, the transcription of the four rhomboid genes identified in Streptomyces coelicolor, the model organism of this genus, was verified by reverse transcription. CONCLUSIONS: Using phylogenetic and genomic analysis, this study demonstrates the existence of five distinct families of rhomboid genes in Streptomycetes. Families A and D are present in all nine species analyzed indicating a potentially important role for these genes. The four rhomboids present in S. coelicolor are transcribed suggesting they could participate in cellular metabolism. Future studies are needed to provide insight into the involvement of rhomboids in Streptomyces physiology. We are currently constructing knock out (KO) mutants for each of the rhomboid genes from S. coelicolor and will compare the phenotypes of the KOs to the wild type strain.