ABSTRACT
PROBLEM: American Indians and Alaska Natives (AIANs) face significant health disparities that are exacerbated by limited access to high-quality, culturally congruent health care providers. There are no premedical postbaccalaureate programs focused on AIAN students. APPROACH: The Northwest Native American Center of Excellence designed the Wy'east Pathway in 2018 to increase the number of AIANs matriculating to U.S. medical schools by supporting those on the cusp of matriculation. Wy'east scholars undertake 10 months of structured programming to augment their academic preparation, improve their Medical College Admission Test (MCAT) scores, and enhance their confidence and cultural identity. Cultural events and mentorship opportunities with AIAN faculty, staff, and cultural liaisons are threaded throughout the pathway curriculum to foster cultural resilience, mentorship, and community. Scholars earn conditional acceptance to Oregon Health and Science University (OHSU) School of Medicine if they complete Wy'east and meet the following criteria: pass all examinations in the primary curricular threads, obtain a qualifying MCAT score, and meet professionalism standards. OUTCOMES: All 14 scholars who successfully completed Wy'east and met criteria in the first 2 cohorts (academic years 2018-2019 and 2019-2020) earned conditional acceptance to OHSU School of Medicine. Ten of the 14 scholars (71.4%) matriculated to OHSU School of Medicine, 2 (14.3%) matriculated to other medical schools, and 2 (14.3%) chose to pursue other health care fields. Wy'east scholars rated the foundational science of medicine and population health and epidemiology threads higher in terms of making them feel better prepared for medical school (mean = 4.71 and 4.83, respectively) than the academic skills and wellness thread (mean = 3.43). NEXT STEPS: Over the next 5 years, Wy'east will grow incrementally to offer a total of 18 conditional acceptance spots per cohort across 3 medical schools. Longitudinal tracking of Wy'east scholars' medical training and career outcomes will be conducted.
Subject(s)
Indians, North American , College Admission Test , Humans , Mentors , Schools, Medical , American Indian or Alaska NativeABSTRACT
Despite extensive efforts to diversify the US physician workforce and increases in both the number of US medical schools and number of students enrolled, there has been no difference in the matriculation and graduation of American Indians and Alaska Natives (AI/AN). Furthermore, advancement remains elusive for AI/AN US medical school faculty, which currently constitutes approximately 0.1% of all US medical school faculty and remains disproportionately underrepresented at the Associate and Full Professor ranks. The Northwest Native American Center of Excellence (NNACoE) aims to address these worrisome trends by implementing innovative programs to support a meaningful journey toward recruiting, training, and supporting AI/AN youth, medical students and faculty. NNACoE has piloted three innovations: 1) Tribal Health Scholars, a 14-week clinical shadowing experience for AI/AN youth in their tribal clinic; 2) Wy'East Post-baccalaureate Pathway, a 9-month structured curriculum with conditional acceptance into Oregon Health & Science University School of Medicine; and 3) Indigenous Faculty Forum, a longitudinal professional development conference for AI/AN medical school faculty to foster career advancement. NNACoE piloted all three programs in 2017 and is actively expanding efforts, while systematically evaluating all programs. Pilot results demonstrate that all Tribal Health Scholars are pursuing college and health science majors, 10 AI/AN Wy'East Post-Baccalaureate Scholars are enrolled to date, and 63 Indigenous medical school faculty are participating in professional development. More systematic evaluation of AI/AN-specific programming is needed to better illuminate how to successfully recruit, train and retain AI/ANs in the US physician workforce.
Subject(s)
/education , Physicians/statistics & numerical data , Workforce/trends , /statistics & numerical data , Humans , Minority Groups/education , Minority Groups/statistics & numerical data , Physicians/supply & distribution , School Admission Criteria , Societies/statistics & numerical data , Societies/trends , Students, Medical/statistics & numerical data , Workforce/statistics & numerical dataABSTRACT
The uncoupling protein (UCP1) is a proton (H+) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H+ gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described. UCP1 is regulated by ß-adrenergic receptors through the sympathetic nervous system and at the molecular activity level by nucleotides and fatty acid to meet thermogenesis needs. The discovery of novel UCP homologs has greatly contributed to the understanding of human diseases, such as obesity and diabetes. In this article, we review the progress made towards the molecular mechanism and function of the UCPs, in particular focusing on the influential contributions from Martin Klingenberg's laboratory. Because all members of the UCP family are potentially promising drug targets, we also present and discuss possible approaches and methods for UCP-related drug discovery.
Subject(s)
Mitochondrial Uncoupling Proteins/chemistry , Mitochondrial Uncoupling Proteins/metabolism , Adenosine Triphosphate/metabolism , Adipose Tissue, Brown/metabolism , Animals , Binding Sites , Fatty Acids, Nonesterified/metabolism , Humans , Protein Binding , Thermogenesis/physiologyABSTRACT
Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.
Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Cell Shape/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Eye Proteins/genetics , Microtubules/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoinositide Phosphatases/genetics , Animals , Cell Differentiation/physiology , Drosophila Proteins/metabolism , Eye/embryology , Phosphoinositide Phosphatases/metabolism , Protein Transport/physiology , Temperature , Vesicular Transport Proteins/metabolismABSTRACT
In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.
Subject(s)
Gene Expression Regulation, Fungal , Mitogen-Activated Protein Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Cycloheximide/pharmacology , Glucose/deficiency , Glucose/pharmacology , Intracellular Signaling Peptides and Proteins , Leupeptins/pharmacology , Membrane Proteins , Mitogen-Activated Protein Kinases/metabolism , Osmotic Pressure , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Kinases , Protein Synthesis Inhibitors/pharmacology , Proteolysis/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Sodium Chloride/pharmacology , Stress, Physiological , Ubiquitination/drug effectsABSTRACT
Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.
Subject(s)
14-3-3 Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Animals , COP-Coated Vesicles/metabolism , COS Cells , Chlorocebus aethiops , HeLa Cells , Humans , Protein Binding , Recombinant Proteins/metabolismABSTRACT
The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region.
Subject(s)
Cell Membrane/metabolism , Golgi Apparatus/metabolism , Phosphatidate Phosphatase/chemistry , Phosphatidate Phosphatase/metabolism , Endoplasmic Reticulum/metabolism , HeLa Cells , Humans , Protein Structure, Tertiary , Protein TransportABSTRACT
Phosphatidylinositol-4-phosphate (PI(4)P) is an important regulator of Golgi function. Metabolic regulation of Golgi PI(4)P requires the lipid phosphatase Sac1 that translocates between endoplasmic reticulum (ER) and Golgi membranes. Localization of Sac1 responds to changes in glucose levels, yet the upstream signaling pathways that regulate Sac1 traffic are unknown. Here, we report that mitogen-activated protein kinase (MAPK) Hog1 transmits glucose signals to the Golgi and regulates localization of Sac1. We find that Hog1 is rapidly activated by both glucose starvation and glucose stimulation, which is independent of the well-characterized response to osmotic stress but requires the upstream element Ssk1 and is controlled by Snf1, the yeast homolog of AMP-activated kinase (AMPK). Elimination of either Hog1 or Snf1 slows glucose-induced translocation of Sac1 lipid phosphatase from the Golgi to the ER and thus delays PI(4)P accumulation at the Golgi. We conclude that a novel cross-talk between the HOG pathway and Snf1/AMPK is required for the metabolic control of lipid signaling at the Golgi.
Subject(s)
Golgi Apparatus/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Endoplasmic Reticulum/metabolism , Glucose/metabolism , Mitogen-Activated Protein Kinases/genetics , Osmotic Pressure , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Transport , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
PtdIns4P is a key regulator of the secretory pathway and plays an essential role in trafficking from the Golgi. Our recent work demonstrated that spatial control of PtdIns4P at the ER (endoplasmic reticulum) and Golgi co-ordinates secretion with cell growth. The central elements of this regulation are specific phosphoinositide 4-kinases and the phosphoinositide phosphatase Sac1. Growth-dependent translocation of Sac1 between the ER and Golgi modulates the levels of PtdIns4P and anterograde traffic at the Golgi. In yeast, this mechanism is largely dependent on the availability of glucose, but our recent results in mammalian cells suggest that Sac1 phosphatases play evolutionarily conserved roles in the growth control of secretion. Sac1 lipid phosphatase plays also an essential role in the spatial control of PtdIns4P at the Golgi complex. A restricted pool of PtdIns4P at the TGN (trans-Golgi network) is required for Golgi integrity and for proper lipid and protein sorting. In mammalian cells, the stress-activated MAPK (mitogen-activated protein kinase) p38 appears to play a critical role in transmitting nutrient signals to the phosphoinositide signalling machinery at the ER and Golgi. These results suggest that temporal and spatial integration of metabolic and lipid signalling networks at the Golgi is required for controlling the secretory pathway.
Subject(s)
Cell Proliferation , Golgi Apparatus/metabolism , Intracellular Membranes/metabolism , Phosphatidylinositols/metabolism , Signal Transduction , Animals , Golgi Apparatus/enzymology , Humans , Membrane Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Transport , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Phosphoinositide lipids were initially discovered as precursors for specific second messengers involved in signal transduction, but have now taken the center stage in controlling many essential processes at virtually every cellular membrane. In particular, phosphoinositides play a critical role in regulating membrane dynamics and vesicular transport. The unique distribution of certain phosphoinositides at specific intracellular membranes makes these molecules uniquely suited to direct organelle-specific trafficking reactions. In this regulatory role, phosphoinositides cooperate specifically with small GTPases from the Arf and Rab families. This review will summarize recent progress in the study of phosphoinositides in membrane trafficking and organellar organization and highlight the particular relevance of these signaling pathways in disease. This article is part of a Special Issue entitled Lipids and Vesicular Transport.
Subject(s)
ADP-Ribosylation Factors/metabolism , Phosphatidylinositols/metabolism , Transport Vesicles/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Cell Membrane/metabolism , Endocytosis , Golgi Apparatus/metabolism , Humans , Intracellular Membranes/metabolism , Lipid Metabolism , Phosphotransferases/metabolism , Protein Transport , Signal TransductionABSTRACT
The protein processing and trafficking function of the Golgi is intimately linked to multiple intracellular signaling pathways. Assembly of Golgi trafficking structures and lipid sorting at the Golgi complex is controlled and coordinated by specific phosphoinositide kinases and phosphatases. The intra-Golgi transport machinery is also regulated by kinases belonging to several functionally distinct families, for example, MAP kinase signaling is required for mitotic disassembly of the Golgi. However, the Golgi plays an additional, prominent role in compartmentalizing other signaling cascades that originate at the plasma membrane or at other organelles. This article summarizes recent advances in our understanding of the signaling network that converges at the Golgi.
Subject(s)
Golgi Apparatus/metabolism , Signal Transduction , Biological Transport , Cell Enlargement , Cell Membrane/metabolism , Cell Proliferation , Cyclic AMP/metabolism , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Lipid Metabolism , Phosphatidylinositols/metabolism , Phosphatidylinositols/physiologyABSTRACT
The enrichment of phosphatidylinositol-4-phosphate (PI(4)P) at the trans Golgi network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here, we show that lipid phosphatase Suppressor of actin mutations 1 (SAC1) is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi-mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase-II (Man-II) and N-acetylglucosamine transferase-I (GnT-I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi-specific defects in N-and O-linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.
Subject(s)
Golgi Apparatus/metabolism , Membrane Proteins/ultrastructure , Phosphatidylinositol Phosphates/metabolism , Protein Transport , Down-Regulation , Glycosylation , Golgi Apparatus/ultrastructure , HeLa Cells , Humans , Membrane Proteins/chemistry , Phosphatidylinositol Phosphates/chemistry , trans-Golgi Network/chemistryABSTRACT
Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved from yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway.
Subject(s)
Golgi Apparatus/metabolism , Phosphatidylinositols/metabolism , Animals , Biological Transport , Cell Proliferation , Humans , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/metabolismABSTRACT
Phosphoinositide lipids play a dual role in cell physiology. Specific sets of these molecules are short-lived downstream mediators of growth signals, regulating cell survival and differentiation. In addition, distinct classes of phosphoinositide lipids function as constitutive mediators of membrane traffic and organelle identity. Recent work has provided the first direct evidence that phosphoinositides also play a direct role in linking protein secretion with cell growth and proliferation. This review focuses on SAC1 lipid phosphatase and how this enzyme operates in an evolutionary conserved mechanism to coordinate the secretory capacity of ER and Golgi during cell growth.
Subject(s)
Lipid Metabolism , Phosphatidate Phosphatase/metabolism , Secretory Pathway , Animals , Cell Membrane/metabolism , Cell Proliferation , Golgi Apparatus/metabolism , Humans , Phosphatidate Phosphatase/chemistry , Phosphatidate Phosphatase/geneticsABSTRACT
When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I-mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor-induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.
Subject(s)
Endoplasmic Reticulum/enzymology , Golgi Apparatus/enzymology , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Animals , COP-Coated Vesicles/metabolism , COP-Coated Vesicles/ultrastructure , COS Cells , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Enzyme Inhibitors/pharmacology , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Mitogens/pharmacology , NIH 3T3 Cells , Protein Transport/physiology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Phosphoinositides play a central role in regulating processes at intracellular membranes. In yeast, a large number of phospholipid biosynthetic enzymes use a common mechanism for transcriptional regulation. Yet, how the expression of genes encoding lipid kinases and phosphatases is regulated remains unknown. RESULTS: Here we show that the expression of lipid phosphatase Sac1p in the yeast Saccharomyces cerevisiae is regulated in response to changes in phosphatidylinositol-4-phosphate (PI(4)P) concentrations. Unlike genes encoding enzymes involved in phospholipid biosynthesis, expression of the SAC1 gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR) of SAC1 that is responsible for PI(4)P-mediated regulation. Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels. CONCLUSION: Regulation of Sac1p expression via the concentration of its major substrate PI(4)P ensures proper maintenance of compartment-specific pools of PI(4)P.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Fungal/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphoric Monoester Hydrolases/biosynthesis , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae/enzymology , 5' Untranslated Regions/genetics , 5' Untranslated Regions/metabolism , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphoric Monoester Hydrolases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Compartment-specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol-4-phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C-terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N-terminal domain is responsible for Golgi localization. We also show that starvation-induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation-induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non-catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth-dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.
Subject(s)
1-Phosphatidylinositol 4-Kinase/physiology , Gene Expression Regulation, Fungal , Golgi Apparatus/metabolism , Phosphoric Monoester Hydrolases/physiology , Saccharomyces cerevisiae Proteins/physiology , Amino Acid Motifs , Biological Transport , Cross-Linking Reagents/chemistry , Endoplasmic Reticulum/metabolism , Genotype , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Models, Biological , Mutagenesis , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Deltasac1 Deltainp54 and sac1ts Deltainp54 mutants. Furthermore, sac1ts Deltainp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Deltasac1 Deltainp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.