Intracellular transport RNDr. Jan Škoda, Ph.D. Department of Experimental Biology ^ Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Outline - Important aspects of comparmentalization - Protein sorting - Protein translocation into nucleus, mitochondria/plastids, ER -Vesicular transport - Secretory and endocytic pathways - Intercellular vesicular transport 2 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Aspects of compartmentalization 3 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Cytosol vs. cytoplasm Cytosol -the fluid contained in the cell without membrane organelles Cytoplasm - Cytosol + membrane organelles without the nucleus Plasma membrane (A) Cytoplasm (B) Cytosol 4 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Prokaryotic vs eukaryotic cells Prokaryotic cells - Most cytosol only (including nucleoid) - Cyanobacteria - thylakoids Eukaryotic cells - Plant cells - usually very limited cytosol (large vacuole) -Cytoplasm full of membrane organelles 5 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Compartmentalization: Pros & Cons PROS ■ Separation of biochemically incompatible processes or potentially harmful processes (degradation, oxidation) - Optimized efficiency - chemically optimal environment (prevents substrate diffusion; different concentration of molecules, e.g., ions; pH) CONS (REQUIREMENTS) ■ Coordination of organelles: biosynthesis, anabolic and catabolic processes (signaling pathways) Transport of molecules between organelles (substrates, precursors, metabolites) ■ Precise sorting of proteins and other molecules 6 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Animal cell Plant cell Fungal cell Plasma membrane Plasma membrane Plasma membrane Glycocalyx Cell wall Cell wall Nucleus Nucleus Nucleus Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum Golgi apparatus Golgi apparatus Golgi apparatus Lysosomes Vacuoles Vacuoles Peroxisomes Peroxisomes Peroxisomes - Glyoxysomes Glyoxysomes Mitochondria Mitochondria Mitochondria - Chloroplasts - Borders of the cell, interaction with its surrounding environment Catabolism Storage and expression of the genetic information, anabolism Energy metabolism, apoptosis 7 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Brief overview of cellular compartments and their function - Cytosol: protein synthesis, signaling and metabolic pathways - Nucleus: genome, DNA and RNA synthesis Endoplasmic reticulum (ER): lipid synthesis, synthesis of secreted and integral membrane proteins, Ca2+ regulation ■ Golgi apparatus: posttranslational modification of proteins, modifications of lipids, cargo sorting to the secretory pathway - Mitochondrion: ATP synthesis (OXPHOS) Chloroplasts: ATP synthesis and carbon fixation (photosynthesis) Lysosomes/vacuoles: degradation of molecules / organelles (autophagy), turgor in plants/fungi Peroxisomes, glyoxysomes: oxidation of long chain fatty acids (p-oxidation), detoxification of various harmful compounds - Membrane vesicles: transport of cargo between organelles/to PM, endosomes, exosomes... 8 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Protein sorting 9 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Protein sorting in eukaryotic cells - Protein synthesis occurs at ribosomes in the cytosol - Exceptions: mitochondrial DNA- and chloroplast DNA-encoded proteins (own ribosomes) - (Nuclear translation under specific stress conditions?) Sorting into specific compartments: -Gated transport: cytosol-nucleus, through nuclear pore complexes -Transmembrane transport: ER, mitochondria, chloroplasts, peroxisomes -Vesiculartransport: ER, Golgi apparatus, endosomes, lysosomes, secretory vesicles, plasma membrane, cell exterior 10 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Overview of protein sorting Co-translational transport Protein translocated during synthesis at ER CYTOSOL NUCLEUS MITOCHONDRIA PEROXISOMES PLASTIDS ENDOPLASMIC RETICULUM GOLGI LATE ENDOSOME EARLY ENDOSOME I T SECRETORY VESICLES Post-translational transport Protein translocated after its complete synthesis in the cytosol KEY: = gated transport = transmembrane transport = vesicular transport CELL EXTERIOR 11 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Targeting proteins into compartments Signal sequence (signal peptide) - 15-60 amino acids long sequence Crucial for targeting to specific organelles/compartments - Different classes of signal sequences - organelle-specific Table 12-3 Some Typical Signal Sequences FUNCTION OF SIGNAL SEQUENCE EXAMPLE OF SIGNAL SEQUENCE Import into nucleus Export from nucleus Import into mitochondria Import into plastid Import into peroxisomes Import into ER Return to ER -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val--Ala- -Lys- -Ala-Gly- -Asp- -+H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-lle-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-fH1IM-Met-Val-Ala-Met-Ala-Met-Ala-Ser-Leu-Gln-Ser-Ser-Met-Ser-Ser-Leu-Ser-Leu-Ser-Ser-Asn-Ser-Phe-Leu-Gly-Gln-Pro-Leu-Ser-Pro-lle-Thr-Leu-Ser-Pro-Phe-Leu-Gln-Gly--Ser-Lys- -COO" +H3N-Met-Met-Ser-Phe-Val-Ser- -Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-GIn--Lys-Asp-Glu- -COO" Some characteristic features of the different classes of signal sequences are highlighted in color. Where they are known to be important for the function of the signal sequence, positively charged amino acids are shown in red and negatively charged amino acids are shown in green. Similarly, important hydrophobic amino acids are shown in white and hydroxylated amino acids are shown in blue. +H3N indicates the N-terminus of a protein; COO- indicates the C-terminus. Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Targeting proteins into compartments -Signal sequence (signal peptide) ■ Often at N-terminus: cleaved by signal peptidase once sorting is complete - Internal signal sequences (e.g., nuclear localization sequence) Protein without a signal sequence remains in the cytosol Genetic engineering - introducing/manipulating the signal sequence: targeting proteins to specific compartments cytosolic protein (no signal sequence) ER protein ER signal sequence (A) NORMAL ER protein with signal sequence removed cytosolic protein with ER signal sequence (B) SWAPPED SIGNAL SEQUENCES 13 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Protein translocation into nucleus 14 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Gated transport: nucleus^cytosol Nuclear pore complexes (NPCs) cytosolic annular fibril subunit CYTOSOL lumenal subunit column subunit outer nuclear membrane ]nuclear envelope NUCLEUS ring subunit ' i nuclear fibril 50 nm B • » • C . 2 8 3 7 % 6 5 m E . — 0.1 IJLIY1 16 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) - 8-fold structure, -30 different proteins, FG-repeats (phenyalanin, glycin rich sequences) - Small molecules (<5 kDa) diffuse freely Macromolecules use nuclear import/export receptors -karyopherins - Import: nuclear proteins, transcription factors, enzymes, etc. - Export: RNA, ribosomal subunits FG-repeats in NPC proteins - Phenyalanin and glycin rich sequences - Important for transport: binding of karyopherins Import via nuclear pore complexes Protein (large macromolecules) must contain nuclear localization sequence (NLS) Importins - karyopherins mediating nuclear import Bind NLS of the transported protein (cargo protein) Bind to and move along FG-repeats of NPC proteins cargo protein 1 cargo protein 2 cargo protein 3 nuclear import receptor ' nuclear import receptor (A) nuclear localization signals nuclear import adaptor protein (B) Importins transport cargo into the nucleus and return to cytosol Needs energy - GTP hydrolysis by GTPase Ran 18 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Ran-regulated transport through NPCs - Ran GTPase in two states: Ran-GTP/Ran-G DP - Ran-GTP/Ran-GDP regulated by: Ran GTPase-activating protein - Ran-GAP, cytosolic Ran guanine nucleotide exchange factor - Ran-GEF, nuclear Ran-GDP Ran-GAP \^ - / - \ CYTOSOL H rCZ Ran-GTP \ / NUCLEUS * 1 IMI 1 1 1 only in the nucleus | -> Cargo unloading 19 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) protein with nuclear localization signal Ran-GDP DISSOCIATES cargo delivered to nucleus NUCLEAR IMPORT Nuclear export through NPCs - rRNA, tRNA, mRNA transcribed in nucleus but active in the cytoplasm; +shuttling proteins Reverse process to nuclear import: Macromolecules must contain nuclear export sequence (NES) - Exportins - karyopherins mediating nuclear export - Needs energy for cargo release 20 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) cargo delivered to cytosol Ran-GDP DISSOCIATES ^ FROM RECEPTORS protein with nuclear export signal NUCLEAR EXPORT Protein translocation into mitochondria and plastids 21 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Transport of proteins into mitochondria and chloroplasts - Protein translocators at outer and inner membranes (+ thylakoid membrane in chloroplasts); Signal sequence at N-terminus Mitochondria Chloroplasts (A) MITOCHONDRION outer membrane matrix space intermembrane space inner membrane (B) CHLOROPLAST outer membrane intermembrane space stroma (matrix space) thylakoid space thylakoid membrane IMS proteins IM proteins ——IMcarrier Matrix proteins TOM complex P-barrelOMP SAM complex IM carrier Stromal thylakoid proteins—s\/—IMS proteins IM proteins (stop transfer) -Mitochondrial Hsp70 TIM23 complex MIA OXA1 TIM22 complex Cytosol complex OM IMS IMS HSP70 ÖjuOiioDüCi TIC complex Matrix/stroma •<— Stromal HSP93 -a-helicalOMP ß-barrelOMP Stromal HSP70 22 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Protein import by mitochondria Proteins transported as unfolded Signal sequence is cleaved by signal peptidase after translocation Final conformation restored by chaperons precursor protein signal sequence INSERTION INTO MEMBRANE BY TOM COMPLEX TOM complex I-1 Insertion into the membrane BINDING TO IMPORT RECEPTORS outer mitochondrial membrane / inner mitochondrial membrane U4- CYTOSOL TIM23 complex receptor protein in TOM complex CLEAVAGE BY SIGNAL PEPTIDASE - Outer membrane: SAM complex (Sorting and assembly machinery) - Inner membrane: TIM or OXA complex (oxidase assembly) TRANSLOCATION INTO MATRIX & MATRIX SPACE mature i mitochondrial 1^ protein cleaved signal peptide 23 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Protein import by mitochondria TOM COMPLEX SAM SPACE chaperones MATRIX SPACE MATRIX SPACE MITOCHONDRIAL PROTEIN SYNTHESIS space chaperones TIM22 COMPLEX protein (C) (D) 24 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Protein translocation into ER 25 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Endoplasmic reticulum Rough ER - Ribosomes at its surface: synthesis of secreted proteins and proteins for ER, Golgi, lysosomes, plasma membrane - Major hub of protein trafficking (vesicular transport) 26 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Protein translocation into ER - Co-translational translocation - close ER-ribosome contact - ER-specific signal sequence navigates attached ribosomes to ER 2 types of proteins: -Water-soluble proteins Fully translocated into the ER lumen; secretory proteins, proteins that function in the lumen of ER or other secretory pathway organelles -Transmembrane proteins - Embedded in the membrane; some function in the ER membrane, most in other organelles, plasma membrane 27 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Rough ER - ribosomes directed by signal sequence - ER and cytosolic ribosomes differ only by the protein they synthesize -SRP (signal-recognition particle) -SRP receptor in rough ER membrane tRNA BINDING OF SRP ,5' TO SIGNAL PEPTIDE CAUSES A PAUSE IN TRANSLATION SRP-BOUND RIBOSOME SRP AND SRP RECEPTOR DISPLACED AND RECYCLED ER LUMEN mRNA encoding a cytosolic protein remains free in cytosol free polyribosome in cytosol 5' I protein translocates SRP receptor protein in rough ER membrane mRNA encoding a polyribosome bound to ER protein targeted membrane by multiple to ER remains nascent polypeptide chains ER membrane membrane-bound 28 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Binding of the signal sequence opens the translocator -Growing polypeptide chain can be translocated through the membrane 29 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Water-soluble vs. Transmembrane proteins CYTOSOL inactive protein active translocator translocator COOH mature soluble protein in ER lumen - Fully into the ER lumen 30 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) t-transfer stop-transfer sequence ER LUMEN hydrophobic hydrophobic stop-transfer- start-transfer- peptide- peptide- | binding site binding site translocator protein 'COOH NH, signal peptidase mature transmembrane protein in ER membrane -Start- and stop-transfer signals Multipass transmembrane proteins - Position of start- and stop-transfer sequences defines protei integration 31 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Vesicular transport - Intracellular transport (6 Apr 2022) Vesicular transport Bidirectional transport among ER-Golgi-plasma membrane Budding and fusion of membrane vesicles Intracellular transport Intercellular communication 33 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Anterograde trafficking nuclear envelope endoplasmic reticulum 9/ ,ov°vQ O O csternae secretory -1 vesicle Golgi apparatus Retrograde trafficking Vesicular road-map of the cell lysosome - Secretory pathway ■ To plasma membrane and endosomes - Endocytic pathway From plasma membrane to endosomes and lysosomes - Retrieval pathway - Returned/reused Golgi apparatus 34 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Transport vesicles - Membrane-bound vesicles -Transport by diffusion (short distance; ER-Golgi) or active transport along cytoskeleton (long distances, directed transport) -Cargo loading - molecules from the lumen & membrane of the donor organelles - from outside the cell & plasma membrane (endocytosis) e.g., membrane lipids and proteins from ER to plasma membrane 35 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl Vesicle budding - Driven by assembly of a protein coat outside the forming vesicle - Different protein complexes (clathrin/adaptins; coatomers) - Clathrin/adaptins - endocytosis, GA to endosomes/lysosomes - COPII (coat protein II) - ER to GA - COPI (coat protein I) - GA to ER -The protein coat is lost after the vesicle forms Main functions of protein coats: - Formation of membranes into vesicles - Cargo selection 36 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Clathrin-coated vesicle formation - Protein coat: network of clathrin triskelions -Specificity: Adaptin binds to cargo receptors - Dynamin rings 37 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Gallery of clathrin cage architectures 28 Mini coat 32 Sweet potato 36 D6 barrel 36 Tennis ball 37 Big apple r\\ 20 Dice 24 Brüssel S # Ä 26 Pumpkin 28 Mini coat 32 Sweet potato (L) 36 Tennis ball 38 Big apple (L) 38 Big apple (R) 38 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Long distance - active transport - Movement along cytoskeleton Microtubule-dependent transport - Actin (microtubule-independent) transport - Motor proteins https://voutu.be/v-uuk4Pr2i8 39 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Target recognition Vesicle tethering - Rab proteins - small G proteins - GTPase activity (Rab-GEF: GDP^ GTP) - GTP form anchored to the membrane - Binds to tethering factors (Rab effectors) at the target membrane Vesicle docking -SNARE - Interaction of v-SNARE (vesicle) & t-SNARE (target) - Affect target specificity by interacting with tethering factors 40 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Target recognition: Rab proteins - SNAREs - fusion \^ J v-SNAR Rab-GTP^ ^TETHERING Rab effector (tethering protein) DOCKING t-SNAREv, I. /, target membrane Adaptor coal prote ns Telhonog factors --» Cytoskeictai track ^ Rab protems JB Motor proieins 41 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Membrane fusion a b Zipping C Hemifusion - SNAREs mediate tight connection between the fusing membranes - Expels water molecules from the hydrophilic surface - Ca2+ generally promotes fusion completion Complex machinery - understanding still limited c 42 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Vesicular transport in brief Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Secretory and endocytic pathways 44 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Secretory pathway -Luminal cargo: secreted proteins, lipids, saccharides - Membrane proteins, lipids/glycolipids: insertion into plasma membrane or function in the vesicles Gradual modifications (maturation) of proteins, lipids - ER - resident luminal enzymes e.g., disulfide bonds between cysteines (stability of secreted proteins) - Protein N-glycosylation (90% glycoproteins) - addition of precursor oligosaccharide in ER -Golgi - membrane bound enzymes - Oligosaccharides processing - Protein glycosylation: O-glycosylation and further N-glycosylation 45 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Glycoconjugation N-Glyc J9} ^ Glycans O-Glycans Glycosamino- GPI Rough "|^r]| ER i— Golgi apparatus |-> r Plasma membrane Glyco- glycans anchors sphingolipids Glc- -Glc in 4, ★Glc- **Glc- ***Glc- * * -Glc í ^ysösome^ ^^^^^^^ /V-LINKED GLYCOSYLATION protein backbone CH, C —O Asparagine N-glycans O-glycans O-LINKED GLYCOSYLATION protein backbone^ H3C —CH Threonine O H /-O \ OH H yl W-acetylglucosamine H NHCOCH3 ,; remainder of oligosaccharide side chain CH2OH hOH H remainder of H NHCOCH3 oligosaccharide side chain ^-acetylgalactosamine Glycosylphosphatidylinositol (GPI) anchors GPI anchor c-9-9 o-c ?.o ER lumen Cytoplasm 46 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Exocytosis / Secretory pathway newly synthesized soluble proteins for constitutive secretion CYTOSOL\ EXTRACELLULAR SPACE newly synthesized plasma membrane lipids unregulated membrane fusion newly synthesized plasma membrane protein Constitutive - Unregulated by stimuli - Operates continuously in all cells Regulated - "On demand" secretion of hormones, neurotransmitters, digestive enzymes in specialized cells - Vesicles wait near plasma membrane - Signal triggers intracellular pathway - often 1^Ca2+ - membrane fusion CONSTITUTIVE SECRETORY PATHWAY -•plasma membrane intracellular signaling pathway V Golgi apparatus secretory vesicle storing secretory proteins regulated membrane fusion signal such as hormone or neurotransmitter 47 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Endocytic pathway - 5 major mechanisms Pinocytosis - general (historic) term Phagocytosis Macropinoeytosis Clathrin- and caveolin-independent pathways Lysosome 48 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Endocytosis - much more complex classification Dynamin dependent- -Dynamin independent- Clathrin FEME Caveolin CLIC/GEEC Macropinocytosis Phagocytosis Early endosome Late endosome Lysosome Reading for interested students 49 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Endocytosis - major differences Clathrin-dependent & caveolin-dependent - Dynamin-dependent -Vesicles <= 100 nm Phagocytosis Macropinocytosis Clathrin- Caveolin-dependent dependent endocytosis endocytosis Clathrin- and caveolin-independent pathways Lysosome Caveolin - Phagocytosis & macropinocytosis - Actin-dependent, dynamin-independent -Vesicles > 200 nm (> 500 nm for phagocytosis) OOJ 50 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Phagocytosis - Recognition of self and non-self - specific receptors on the surface - Conserved among vertebrates and invertebrates - Defense against infections (immune system) - e.g., macrophages or hemocvtes - Food uptake - protists - Actin-mediated formation of pseudopodia - Large particle enclosed in the phagosome (vesicle) - fusion with (maturation into) lysosome 51 Bi1700en Cell Biology/05-Intracellular transport (6 Apr 2022) I Lysosomes - Highly acidic pH - 5 maintained by V-ATPase proton pumps - Important for acidic hydrolases (-50 different) Not active in the cytosol due to pH - Heavily glycosylated membrane proteins are protected from hydrolases Fuse with and degrade cargo of - Endosomes (general endocytic pathway) - Phagosomes (phagocytosis) - Autophagosomes (autophagy - self degradation) 0.2-0.5 |xm CYTOSOL 52 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Pathways leading to degradation in EXTRACELLULAR FLUID \ I_ 1 fxm 53 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Intercellular vesicular transport 54 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Intercellular vesicular transport Exosomes - Derived from multivesicular bodies (MVB or MVE) -Generally smaller: 30-100 nm Microvesicles - Plasma membrane budding and fission -Larger: 100-1000 nm (^) Microvesicles Exosomes a 55 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) Formation of multivesicular bodies early endosome multivesicular body Rerouted to plasma membrane: exosomes Fusion with lysosome: degradation of membrane proteins and lipids 56 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022) fl SARS-COV-2 clathrin-mediated endocytosis -Viral S protein binds to ACE2 receptor on the cell surface 57 Bi1700en Cell Biology / 05 - Intracellular transport (6 Apr 2022)