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Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Recognize amino acids by their single letter codes. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Recognize amino acids by their single letter codes. Identify positive, negative or hydrophobic amino acid residues in a protein sequence. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Recognize amino acids by their single letter codes. Identify positive, negative or hydrophobic amino acid residues in a protein sequence. Recognize patterns of amino acid residues that serve as signals to target proteins to subcellular locations. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Recognize amino acids by their single letter codes. Identify positive, negative or hydrophobic amino acid residues in a protein sequence. Recognize patterns of amino acid residues that serve as signals to target proteins to subcellular locations. Use amino acid sequence information to identify a protein in the NCBI data bases. Objectives: Be familiar with the various subcellular compartments in eucaryotic cells. Know types of proteins that would be found in the different subcellular compartments. Use PubMed to find an article about proteins present in bacteria Recognize amino acids by their single letter codes. Identify positive, negative or hydrophobic amino acid residues in a protein sequence. Recognize patterns of amino acid residues that serve as signals to target proteins to subcellular locations. Use amino acid sequence information to identify a protein in the NCBI data bases. Use computational tools to predict the subcellular location for a protein of given sequence (homework) M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? What would be the subcellular location of a protein with this sequence of amino acids? M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? What would be the subcellular location of a protein with this sequence of amino acids? How would such a protein be delivered to its final location? M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? What would be the subcellular location of a protein with this sequence of amino acids? How would such a protein be delivered to its final location? First lets review the possible locations in a cell ---> ----> Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. • Translation of the mRNA on ribosomes on rough Endoplasmic Reticulum (ER) to make the protein. Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. • Translation of the mRNA on ribosomes on rough Endoplasmic Reticulum (ER) to make the protein. •The protein is threaded into the lumen of the ER because of signal sequence of amino acids (blue) near amino terminus of the protein. Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. • Translation of the mRNA on ribosomes on rough Endoplasmic Reticulum (ER) to make the protein. •The protein is threaded into the lumen of the ER because of signal sequence of amino acids (blue) near amino terminus of the protein. •The protein is passed on to the Golgi. Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. • Translation of the mRNA on ribosomes on rough Endoplasmic Reticulum (ER) to make the protein. •The protein is threaded into the lumen of the ER because of signal sequence of amino acids (blue) near amino terminus of the protein. •The protein is passed on to the Golgi. •The protein is enclosed in a membrane vesicle which leaves the Golgi and takes it to the Plasma Membrane (PM) Pathway to secretion of the protein to the outside of the cell. For example, secretion of a digestive enzyme such a lipase from a cell in the pancreas. • Transcription of the mRNA that codes for the protein from DNA in the nucleus. • Export of the mRNA from the nucleus through pores in the nuclear envelope. • Translation of the mRNA on ribosomes on rough Endoplasmic Reticulum (ER) to make the protein. •The protein is threaded into the lumen of the ER because of signal sequence of amino acids (blue) near amino terminus of the protein. •The protein is passed on to the Golgi. •The protein is enclosed in a membrane vesicle which leaves the Golgi and takes it to the Plasma Membrane (PM) •The membrane of the vesicle fuses with the PM releasing the protein to the outside of the cell (eg., lipase secreted from pancreatic cells) Figure 7.16 Review: relationships among organelles of the endomembrane system Proteins that follow this ER/Golgi pathway can also go to Plasma Membrane, eg. Integrins Integrins are proteins that recognize other cells, cause cells to stick together. Human diseases result from defects in integrin genes. A defect in integrin beta3 causes prolonged bleeding, because blood plateletes can’t stick together. Glanzman's Thrombasthenia. With defects in either alpha6 or beta4 integrin skin cells cannot stick together well. Patients are born with blistering epidermis and also have blisters within the mouth and digestive tract...depending on the severity of the disease. Some die within days and others live. Junctional epidermolysis bullosa Proteins that follow this ER/Golgi pathway can also go to Plasma Membrane, eg. Integrins Integrins are proteins that recognize other cells, cause cells to stick together. Lysosomes, Hydrolases. Hydrolases are digestive enzymes that use water to break apart molecules such as proteins, DNA, lipids, polysaccharides. Proteins that follow this ER/Golgi pathway can also go to Plasma Membrane, eg. Integrins Integrins are proteins that recognize other cells, cause cells to stick together. Lysosomes, Hydrolases. Hydrolases are digestive enzymes that use water to break apart molecules such as proteins, DNA, lipids, polysaccharides. Defects in lysosomal genes result in “storage diseases” If a hydrolase is defective the molecules it digests accumulate in lysosomes. Other proteins are translated from their respective mRNA’s in the cytosol and then delivered to different subcellular locations: Mitochondria Peroxisomes Chloroplasts (in plant cells) Nucleus Or some remain in the cytosol What types of proteins go to these different locations and what information directs them to those locations? Mitochondria - e.g., Dehydrogenases Peroxisomes - e.g., Oxidases Chloroplasts (in plant cells) - proteins of photosynthesis Nucleus - e.g., proteins that replicate DNA or regulate genes Cytosol - e.g., enzymes that metabolize glucose Do all cells have all these different proteins and subcellular compartments? Eucaryotes Animals, flies, worms, yeast cells have these compartments and many proteins that are homologous. Do all cells have all these different proteins and subcellular compartments? Eucaryotes Animals, flies, worms, yeast cells have these compartments and many proteins that are homologous. Plant cells have all the compartments plus chloroplasts and a central vacuole. Do all cells have all these different proteins and subcellular compartments? Eucaryotes Animals, flies, worms, yeast cells have these compartments and many proteins that are homologous. Plant cells have all the compartments plus chloroplasts and a central vacuole. Procaryotes Bacterial cells do not have the compartments and have fewer genes, fewer proteins. Do all cells have all these different proteins and subcellular compartments? Eucaryotes Animals, flies, worms, yeast cells have these compartments and many proteins that are homologous. Plant cells have all the compartments plus chloroplasts and a central vacuole. Procaryotes Bacterial cells do not have the compartments and have fewer genes, fewer proteins. Each cell of an organism has DNA that encodes all the possible genes for that organism. Are all the possible proteins present in every cell of the organism? Questions about the genome in an organism: How much DNA, how many nucleotides? How many genes are there? What types of proteins appear to be coded by these genes? Questions about the proteome: What proteins are present? Where are they? When are they present - under what conditions? Questions about the proteome: What proteins are present? Where are they? When are they present - under what conditions? What other proteins and molecules does each protein interact with? It depends on the type of cell, bacteria, yeast, worm, fly, plant, human Animal cell - is a EUCARYOTE Animal cell - is a EUCARYOTE - has a nucleus and other membrane enclosed subcellular compartments, mitochondria, peroxisomes, etc. Plant cell - is also a EUCARYOTE - has a nucleus, mitochondria, peroxisomes, plus chloroplasts, central vacuole. Vibrio cholerae causes cholera ATP drive motor protein complex E. Coli - normal inhabitant of human gut Bacterial cells - are PROCARYOTES Vibrio cholerae causes cholera ATP drive motor protein complex E. Coli - normal inhabitant of human gut Bacterial cells - are PROCARYOTES - NO nucleus, NO membrane enclosed subcellular compartments, NO mitochondria, NO peroxisomes, etc. E. coli genome • 4,639,221 nucleotide pairs • Protein-coding genes yellow or orange bars • genes coding only RNA green arrows What are all the different types of RNAs? Organism . H. influenzae (bacterium) Genome size (Megabases, 10^6) 1.8 Mb Estimated number of genes 1700 Type of Organism Procaryote, no nucleus . Organism . Genome size (Megabases, 10^6) Estimated number of genes Type of Organism H. influenzae (bacterium) 1.8 Mb 1700 Procaryote, no nucleus S. cerevisae (Yeast) 12 Mb 6000 Eucaryote, Unicellular . Organism . Genome size (Megabases, 10^6) Estimated number of genes Type of Organism . H. influenzae (bacterium) 1.8 Mb 1700 Procaryote, no nucleus S. cerevisae (Yeast) 12 Mb 6000 Eucaryote, Unicellular C. elegans (nematode worm) 97 Mb 19,000 Eucaryote, Multicellular Organism . Genome size (Megabases, 10^6) Estimated number of genes Type of Organism . H. influenzae (bacterium) 1.8 Mb 1700 Procaryote, no nucleus S. cerevisae (Yeast) 12 Mb 6000 Eucaryote, Unicellular C. elegans (nematode worm) 97 Mb 19,000 Eucaryote, Multicellular Arabidopsis (plant) 100 Mb 25,000 Eucaryote Multicellular Organism . Genome size (Megabases, 10^6) Estimated number of genes Type of Organism . H. influenzae (bacterium) 1.8 Mb 1700 Procaryote, no nucleus S. cerevisae (Yeast) 12 Mb 6000 Eucaryote, Unicellular C. elegans (nematode worm) 97 Mb 19,000 Eucaryote, Multicellular Arabidopsis (plant) 100 Mb 25,000 Eucaryote Multicellular Drosophila (fruit fly) 180 Mb 13,000 Eucaryote Multicellular Organism . Genome size (Megabases, 10^6) Estimated number of genes Type of Organism . H. influenzae (bacterium) 1.8 Mb 1700 Procaryote, no nucleus S. cerevisae (Yeast) 12 Mb 6000 Eucaryote, Unicellular C. elegans (nematode worm) 97 Mb 19,000 Eucaryote, Multicellular Arabidopsis (plant) 100 Mb 25,000 Eucaryote Multicellular Drosophila (fruit fly) 180 Mb 13,000 Eucaryote Multicellular Homo sapiens (human) 3200 Mb 40,000 Eucaryote Multicellular Are all the genes in a cell producing proteins? Egg cell genes determine nature of whole multicellular organism. Sea urchin egg gives rise to a sea urchin. (A, B) Mouse egg gives rise to a mouse. (C,D) The different types of cells look like they would have different proteins - hair, eyes, spines, etc. How do sizes of egg cells compare to E.coli? Each cell contains a fixed set of DNA molecules—its archive of genetic information. Each cell contains a fixed set of DNA molecules—its archive of genetic information. A given segment of this DNA serves to guide the synthesis of many identical RNA transcripts, which serve as working copies of the information stored in the archive. Each cell contains a fixed set of DNA molecules—its archive of genetic information. A given segment of this DNA serves to guide the synthesis of many identical RNA transcripts, which serve as working copies of the information stored in the archive. Many different sets of RNA molecules can be made by transcribing selected parts of a long DNA sequence, allowing each cell to use its information store differently. Do cells produce proteins from all their genes? What technique can be used to find out? Open Netscape or Explorer. Go to PubMed at http://www.ncbi.nih.gov/entrez/query.fcgi Search PubMed for Slonczewski JL Pairs of students work together: What type of cell are they working on? What question are they trying to answer? What techinques are they using? Open one of the figures. Tell everyone how to find that figure. Explain what is seen in that figure. How were the proteins identified? What were the conclusions? Kirkpatrick C, Maurer LM, Oyelakin NE, Yoncheva YN, Maurer R, Slonczewski JL. Acetate and formate stress: opposite responses in the proteome of Escherichia coli. J Bacteriol. 2001 Nov;183(21):6466-77. Blankenhorn D, Phillips J, Slonczewski JL. Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis. J Bacteriol. 1999 Apr;181(7):2209-16. Genome Proteins for: Metabolism, energy E. coli (bacteria) Procaryote No nucleus or organelles Saccharomyces (Yeast) Eucaryote, single cells Nucleus, Mitochondria, ER, Golgi, peroxisomes 4,640,000 12,050,000 nucleotides 890 820 Genome E. coli (bacteria) Procaryote No nucleus or organelles Saccharomyces (Yeast) Eucaryote, single cells Nucleus, Mitochondria, ER, Golgi, peroxisomes 4,640,000 12,050,000 nucleotides Proteins for: Metabolism, energy DNA replication, repair Transcription of RNA 890 120 230 820 175 400 Genome E. coli (bacteria) Procaryote No nucleus or organelles Saccharomyces (Yeast) Eucaryote, single cells Nucleus, Mitochondria, ER, Golgi, peroxisomes 4,640,000 12,050,000 nucleotides Proteins for: Metabolism, energy DNA replication, repair Transcription of RNA Translation Cell Structure Protein targeting, secretion 890 120 230 180 180 35 820 175 400 350 250 430 Genome E. coli (bacteria) Procaryote No nucleus or organelles Saccharomyces (Yeast) Eucaryote, single cells Nucleus, Mitochondria, ER, Golgi, peroxisomes 4,640,000 12,050,000 nucleotides Proteins for: Metabolism, energy DNA replication, repair Transcription of RNA Translation Cell Structure Protein targeting, secretion 890 120 230 180 180 35 820 175 400 350 250 430 Does E.coli produce all proteins constantly, or selected ones? Where are the proteins located in the yeast cell? This is the sequence of amino acids at the end of a protein that is targeted to a certain subcellular compartment. M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? This is the sequence of amino acids at the end of a protein that is targeted to a certain subcellular compartment. M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? What would be the subcellular location of a protein with this sequence of amino acids? This is the sequence of amino acids at the end of a protein that is targeted to a certain subcellular compartment. M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QWhat does this mean in the language of proteins? What would be the subcellular location of a protein with this sequence of amino acids? How would such a protein be delivered to its final location? What are the functions of protein in this subcellular location? To understand the information in proteins that targets them to the respective subcellular compartments you need to be able read amino acid sequences. Also, amino acid sequences can indicate the function of the protein. You need to recognize the amino acids by their single letter abbreviations. Recognize those that are non-polar, hydrophobic. Recognize the polar, hydrophyllic ones. Recognize the charged ones, positive or negative. You need to recognize the amino acids by their single letter abbreviations. Recognize those that are non-polar, hydrophobic. Recognize the polar, hydrophyllic ones. Recognize the charged ones, positive or negative. Glycine is the simplest amino acid. You need to recognize the amino acids by their single letter abbreviations. Recognize those that are non-polar, hydrophobic. Recognize the polar, hydrophyllic ones. Recognize the charged ones, positive or negative. Glycine is the simplest amino acid. Its single letter abbreviation is G G G A G A V Hydrophobic L I G A V Hydrophobic M L I G A V L I Hydrophobic M F W P S T Hydrophyllic S T C Hydrophyllic Y N Q S T C Hydrophyllic D E Y N Q S T C Y N Q R H Hydrophyllic D E K Figure 5.16 Making a polypeptide chain Figure 5.16 Making a polypeptide chain amino end carboxyl end Figure 5.16 Making a polypeptide chain Figure 5.16 Making a polypeptide chain What are the names of these amino acid residues? Figure 5.18 The primary structure of a protein Figure 5.20 The secondary structure of a protein Figure 5.17 Conformation of a protein, the enzyme Lysozyme Figure 5.19 A single amino acid substitution in a protein causes sickle-cell disease A change in one amino acid can change the structure and function of a protein What chemical difference between Glu and Val? Figure 5.19 A single amino acid substitution in a protein causes sickle-cell disease A change in one amino acid can change the structure and function of a protein What chemical difference between Glu and Val? Some Typical Signal Sequences that direct proteins to different subcellular compartments Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q- Retention in lumen of ER -K-D-E-L Some Typical Signal Sequences that direct proteins to different subcellular compartments Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q- Retention in lumen of ER -K-D-E-L Import into mitochondria M-L-S-L-R-Q-S-I-R-F-F-K-P-A-T-R-T-L-C-S-S-R-Y-L-L- Some Typical Signal Sequences that direct proteins to different subcellular compartments Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q- Retention in lumen of ER -K-D-E-L Import into mitochondria M-L-S-L-R-Q-S-I-R-F-F-K-P-A-T-R-T-L-C-S-S-R-Y-L-L- Import into nucleus -P-P-K-K-K-R-K-V- Import into peroxisomes -S-K-L Some Typical Signal Sequences Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QRetention in lumen of ER -K-D-E-L Import into mitochondria M-L-S-L-R-Q-S-I-R-F-F-K-P-A-T-R-T-L-C-S-S-R-Y-L-LImport into nucleus -P-P-K-K-K-R-K-V- Import into peroxisomes -S-K-L An extended block of hydrophobic amino acids is shown in blue. The amino terminus of a protein is toward the left; the carboxyl terminus to the right. Which ones are positively or negatively charged?? Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QA hydrophobic series near the amino terminus Retention in lumen of ER At the carboxyl terminus -K-D-E-L Import into mitochondria M-L-S-L-R-Q-S-I-R-F-F-K-P-A-T-R-T-L-C-S-S-R-Y-L-LRegularly spaced positive residues near amino terminus Import into nucleus -P-P-K-K-K-R-K-VA patch of positive residues in the middle Import into peroxisomes -S-K-L A small residue, positive residue, hydrophobic at carboxyl Positively charged amino acids are shown in green, and negatively charged amino acids in red. An extended block of hydrophobic amino acids is shown in blue. The amino terminus of a protein is toward the left; the carboxyl terminus to the right. What is this protein? Where is it located? http://www.ncbi.nlm.nih.gov/blast/ use the Standard Protein-Protein Blast Enter the amino acid sequence and submit it to the Blast search >P11310 MAAGFGRCCRVLRSISRFHWRSQHTKANRQREPGLGFSFEFTEQQKEFQATARKF AREEIIPVAAEYDKTGEYPVPLIRRAWELGLMNTHIPENCGGLGLGTFDACLISEE LAYGCTGVQTAIEGNSLGQMPIIIAGNDQQKKKYLGRMTEEPLMCAYCVTEPGA GSDVAGIKTKAEKKGDEYIINGQKMWITNGGKANWYFLLARSDPDPKAPANKA FTGFIVEADTPGIQIGRKELNMGQRCSDTRGIVFEDVKVPKENVLIGDGAGFKVA MGAFDKTRPVVAAGAVGLAQRALDEATKYALERKTFGKLLVEHQAISFMLAEM AMKVELARMSYQRAAWEVDSGRRNTYYASIAKAFAGDIANQLATDAVQILGGN GFNTEYPVEKLMRDAKIYQIYEGTSQIQRLIVAREHIDKYKN >P11310 is ACYL-COA DEHYDROGENASE, MEDIUM-CHAIN SPECIFIC PRECURSOR It is delivered to the Mitochondria What is this protein? http://www.ncbi.nlm.nih.gov/blast/ use the Standard Protein-Protein Blast Enter the amino acid sequence and submit it to the Blast search >gi|17560134|ref|NP_508036.1 MNRYICEGDNPDITEERKKASFNVDKLTEYYYGGEKRLKARREVEKCVEDHKELQD LKPTPFMSRDELIDNSVRKLAGMAKNYKMIDLTNIEKTTYFLQLVHVRDSMAFSLHY LMFLPVLQSQASPEQLAEWMPRALSGTIIGTYAQTEMGHGTNLSKLETTATYGQKTS EFVLHTPTISGAKWWPGSLGKFCNFAIIVANLWTNGVCVGPHPFLVQIRDLKTHKTLP NIKLGDIGPKLGSNGSDNGYLVFTNYRISRGNMLMRHSKVHPDGTYQKPPHSKLAYG GMVFVRSMMVRDIANYLANAVTIATRYSTVRRQGEPLPGAGEVKILDYQTQQYRILP YIAKTIAFRMAGEELQQAFLNISKDLRQGNASLLPDLHSLSSGLKAVVTFEVQQGIEQ CRLACGGHGYSHASGIPELSAFSCGSCTYEGDNIVLLLQVANECELYPEHEAWNRCSI ELCKAARWHVRLYIVRNFLQKVCTAPKDLQPVLRALSNLYIFDLQVSNKGHFMENG YMTSQQIDQLKMGINESLSTIRPDAVSIVDGFAIHEFELKSVLGRRDGNVYPGLFEWT KHSQLNNKEVHPAFDKYLTPIMDKIRAKM gi|17560134|ref|NP_508036.1 is Acyl-Coenzyme A oxidase peroxisomal like family member from the nematode worm [Caenorhabditis elegans]. It is targeted to Peroxisomes by the three amino acids at its carboxyl terminus The typical Signal Sequences that direct proteins to different subcellular compartments Import into ER M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-QA hydrophobic series near the amino terminus Retention in lumen of ER At the carboxyl terminus -K-D-E-L Import into mitochondria M-L-S-L-R-Q-S-I-R-F-F-K-P-A-T-R-T-L-C-S-S-R-Y-L-LRegularly spaced positive residues near amino terminus Import into nucleus -P-P-K-K-K-R-K-VA patch of positive residues in the middle Import into peroxisomes -S-K-L or A-K-M or similar, A small residue, positive residue, hydrophobic at carboxyl What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells)? What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells) - proteins of photosynthesis What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells) - proteins of photosynthesis Nucleus ? ? What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells) - proteins of photosynthesis Nucleus - e.g., proteins that replicate DNA or regulate genes, transcription factors What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells) - proteins of photosynthesis Nucleus - e.g., proteins that replicate DNA or regulate genes, transcription factors Cytosol - e.g., enzymes that metabolize glucose Mitochondria - e.g., Dehydrogenases, metabolism to obtain energy What are the functions of the proteins that are targeted to the different subcellular locations? ER/Golgi pathway Secreted proteins - e.g., pancreatic digestive enzymes, proteases such as trypsin Lysosomal enzymes - e.g., acid hydrolases such as acid proteases, lipases, DNAases, etc. Plasma Membrane proteins - e.g., Integrins. Chloroplasts (in plant cells) - proteins of photosynthesis Nucleus - e.g., proteins that replicate DNA or regulate genes, transcription factors Cytosol - e.g., enzymes that metabolize glucose Mitochondria - e.g., Dehydrogenases, metabolism to obtain energy Peroxisomes - e.g., Oxidases, metabolism when energy is not needed Figure 7.9 The nucleus and its envelope Figure 7.x1 Nuclei and F-actin in BPAEC cells Figure 7.17 The mitochondrion, site of cellular respiration Figure 7.19 Peroxisomes A comparison of a mitochondrial dehydrogenase to a peroxisomal oxidase, both of which metabolize fat. H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H A Fatty acid, which can be oxidized in mitochondria or peroxisomes Dehydrogenase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Dehydrogenase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Dehydrogenase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Dehydrogenase H H 2H’s H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Creates a double bond. Dehydrogenase 2H’s H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Creates a double bond. Dehydrogenase 2H’s H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H In mitochondria a Dehydrogenase takes two Hydrogens (2H’s) from the fatty acid. Creates a double bond. Dehydrogenase 2H’s NAD H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Creates a double bond. Dehydrogenase 2H’s NAD H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Creates a double bond. Dehydrogenase NADH2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Creates a double bond. Dehydrogenase NADH2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Dehydrogenase NADH2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Dehydrogenase NADH2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The mitochondrial Dehydrogenase transfers the two Hydrogens (2H’s) to NAD. Dehydrogenase NADH2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H ATP The Hydrogens are delivered to the inner membrane to make ATP Oxidase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H In Peroxisomes an Oxidase takes two Hydrogens (2H’s) from a fatty acid. Oxidase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H Oxidase H H H H H H H H H-C-C-C-C-C-C-C-C-COOH H H H H H H H H Oxidase H H 2H’s H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase 2H’s H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase 2H’s H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase 2H’s O2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase 2H’s O2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase H2 O2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H Oxidase H2 O2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The Peroxisomal Oxidase transfers the two Hydrogens (2H’s) to Oxygen to make Hydrogen Peroxide (H2O2). Oxidase H2 O2 H H H H H H H H-C-C-C-C-C=C-C-C-COOH H H H H H H H The Peroxisomal Oxidase transfers the two Hydrogens (2H’s) to Oxygen to make Hydrogen Peroxide (H2O2) No ATP is made in Peroxisomes What happens if there is a genetic defect in a peroxisomal protein? If one of the nucleotides is changed in the gene, in the DNA, then an amino acid may be changed and the resulting protein may no longer function. What happens if there is a genetic defect in a peroxisomal protein? If one of the nucleotides is changed in the gene, in the DNA, then an amino acid may be changed and the resulting protein may no longer function. If the protein is a fatty acid oxidase, then unmetabolized fatty acid will accumulate, damage nervous system, and result in mental degeneration after several years of life Adrenoleukodystrophy (Lorenzo’s Oil). What happens if there is a genetic defect in a peroxisomal protein? If one of the nucleotides is changed in the gene, in the DNA, then an amino acid may be changed and the resulting protein may no longer function. If the protein is a fatty acid oxidase, then unmetabolized fatty acid will accumulate, damage nervous system, and result in mental degeneration after several years of life Adrenoleukodystrophy (Lorenzo’s Oil). If the protein is the receptor that recognizes the Signal Sequence (-SKL) then most proteins will not be imported into peroxisomes. Infant does not survive - Zellwegers Syndrome. Each protein contains much information, to be recognized by other proteins, to recognize the molecules it acts on. The specific functions of a cell depend on groups of proteins interacting with each other and with other molecules, DNA, small molecules. To understand these complex interactions computational tools can be employed to predict the functions of individual proteins and groups of proteins. The Howard Hughes Medical Institute (HHMI) is supporting the GWU program to involve undergraduate students in research that involves computational approaches to biological problems. New insights on biological functions and disease will come from researchers and doctors who know biology and computational tools. The HHMI program has funds to support summer undergraduate research internships, new computer science courses for biologists, new courses where biology and computer science students will work together to investigate biological problems.