Protein Sorting in Eukaryotic Cells (Lecture Notes) PDF

Protein sorting Model of nuclear pore Model of a nuclear pore Learning Objectives: Understand the mechanisms by which proteins are targeted to different cellular compartments Understand the concept of signal sequences Understand how the Ran- Understand GTPase cyclehowlead...

Protein sorting Model of nuclear pore Model of a nuclear pore Learning Objectives: Understand the mechanisms by which proteins are targeted to different cellular compartments Understand the concept of signal sequences Understand how the Ran- Understand GTPase cyclehowleads to proteins are targeted to mitochondria and chloroplasts post- nuclear import of proteins translationally Understand the mechanism by which SRP couples translation and translocation of proteins into the endoplasmic reticulum Understand how proteins that have many transmembrane regions are inserted into the endoplasmic reticulum Understand protein glycosylation and folding in the ER  Eukaryotic cells have membrane-bound organelles plasma membrane Figure 1-24A Essential Cell Biology  Proteins are sorted by several mechanisms Most proteins remain In the cytosol cytosol Figure 15-5 Essential Cell Biology Today’s topics 1.“Signal” (“sorting”) sequences (aka “sorting sign 2.Transport into the nucleus 3.Transport into mitochondria 4.Transport into the endoplasmic reticulum 5.Intro to vesicle transport  Signal sequences are necessary to direct a protein to its destination NH2- - COOH Signal sequence(see Table 15-3 for examples) Figure 15-6 Essential Cell Biology  Signal sequences are necessary and sufficient to direct a protein to its destination NH2- - COOH Signal sequence Figure 15-6 Essential Cell Biology We use signal sequences to target GFP to organelles for fluorescence microscopy Signal Sequence for ER GFP NH2- - COOH Part of a cell showing the ER network by fluorescence microscopy Today’s topics 1.“Signal” ( “sorting”) sequences 2.Transport into the nucleus 3.Transport into mitochondria 4.Transport into the endoplasmic reticulum 5.Intro to vesicle transport  Proteins are sorted by several mechanisms cytosol Figure 15-5 Essential Cell Biology  Architecture of the nuclear envelope What types of molecules have to get out of the nucleus? mRNAs, tRNAs, ribosome subunits What types of molecules have to get into the nucleus? Many proteins that interact with the genome, like DNA polymerase, RNA polymerase, transcription factors Figure 15-7 Essential Cell Biology  Schematic of nuclear pore complexes Unstructured protein loops create a diffusion barrier Very small proteins (about 5 kD) can passively diffuse through nuclear po but most proteins do not. Many bigger proteins do enter the nucleus through these pores… but they require an active process of shuttling! Figure 15-8a Essential Cell Biology  The Structure of a nuclear pore complex side view cryo-EM and AI-based structure predictions (Mosalaganti et al. 2022) Nuclear transport receptors move molecules through (‘NLS’) But how does the import receptor ‘let go’ of the cargo protein? Figure 15-9 Essential Cell Biology  Small GTP-binding proteins are molecular switches (aka “GTPases” or ”monomeric GTPases”) GUANINE GDP “GEF” Guanine GTP hydrolysis nucleotide “GAP” Exchange GTPase Activating Factor Protein GTP binding GUANINE GTP bind “effector” proteins to activate a downstream pathway TP binding protein and GTP hydrolysis drive nuclear tr Figure 15-10 Essential Cell Biology TP binding protein and GTP hydrolysis drive nuclear tr Ran GTPase competes with cargo for binding to the receptor Figure 15-10 Essential Cell Biology TP binding protein and GTP hydrolysis drive nuclear tr Figure 15-10 Essential Cell Biology TP binding protein and GTP hydrolysis drive nuclear tr eptor binds to Ran when Ran binds GTP, but not when Ran binds Figure 15-10 Essential Cell Biology TP binding protein and GTP hydrolysis drive nuclear tr import requires high Ran-GTP in the nucleus, and GTP hydrolysis in the Figure 15-10 Essential Cell Biology  Ran GTP hydrolysis and GDP/GTP exchange is controlled by other proteins: a ‘GEF’ and a ‘GAP’ P = GTPase Activating Protein Associated with cytosolic fibrils of nuclear pore Associated with nuclear chromatin (DNA) GEF = Guanine nucleotide Exchange F Figure 12-10 Essential Cell Biology PollEV question: Which of the following would occur if you depleted the cell of Ran GAP? A Ran would accumulate in the nucleus bound to the nuclear transport receptor. B Ran would accumulate in the cytosol bound to the nuclear transport receptor. YES! C The nuclear transport receptor would accumulate in the nucleus and Ran in the cytosol. D The nuclear transport receptor would accumulate in the cytosol and Ran in the nucleus. E None of the above. Today’s topics 1.“Signal” ( “sorting”) sequences 2.Transport into the nucleus 3.Transport into mitochondria (and chloroplasts) 4.Transport into the endoplasmic reticulum 5.Intro to vesicle transport  Proteins are sorted by several mechanisms cytosol Figure 15-5 Essential Cell Biology  Mitochondria Outer membrane Inner membrane A section through a mitochondrion visualized by electron microscopy Figure 1-18 Essential Cell Biology ECB movie 15-2  Mitochondrial protein import Most mitochondrial proteins are encoded in the nuclear genome! Protein is unfolded as it is translocated Protein is translocated across both membranes simultaneously Protein is synthesized and folds in cytosol ….and refolded inside the mitochondrion ort of proteins into chloroplasts occurs through a very similar pr Figure 15-11 Essential Cell Biology Today’s topics 1.“Signal” ( “sorting”) sequences 2.Transport into the nucleus 3.Transport into mitochondria (and chloroplasts) 4.Transport into the endoplasmic reticulum 4. Intro to vesicle transport  Proteins are sorted by several mechanisms cytosol Figure 15-5 Essential Cell Biology The ER is the site of synthesis for secreted proteins and for membrane and lumenal proteins of the ER, Golgi, endosomes, lysosomes, and plasma membrane ~30% of genes! Lumenal protein Membrane protein Golgi Apparatus Endosome and Lysosome Plasma membrane and secreted proteins  An ER Signal Sequence and ‘SRP’ directs a ribosome to the endoplasmic reticulum Watch Movie 15.4 in ECB6! 1. SRP binds signal 2. SRP binds 3. SRP leaves and 4. Translation sequence and SRP receptor on ribosome engages resumes slows/pauses ER translocation translocating translation channel protein across (aka bilayer “translocon”) aka “translocon” “co-translational translocation”  An ER Signal Sequence and ‘SRP’ directs a ribosome to the endoplasmic reticulum Watch Movie 15.4 in ECB6! 1. SRP binds signal 2. SRP binds 3. SRP leaves and 4. Translation sequence and SRP receptor on ribosome engages resumes slows/pauses ER translocation translocating translation channel protein across Translation by the ribosome provides thebilayer driving force needed to move the protein across the membrane aka “translocon” “co-translational translocation”  N-terminal signal sequences are cleaved (removed) on the lumenal side e so m of the ER membrane i bo R t n! o n o w sh and then the protein folds The signal sequence is cleaved off by ‘signal peptidase’ in the ER lumen… Figure 15-15 Essential Cell Biology  Transmembrane proteins are integrated into the ER membrane om If there isduring translocation more than one signal sequence in a s i bo d protein, the first is called a “start-transfer” R an ot e P n n! sequence and the second signal sequence is SR ow called a “stop-transfer” sequence sh Notice, this mechanism defines the orientation of the protein in the membrane: N-terminus in the lumen…. Figure 15-16 Essential Cell Biology rnal signal sequences are not cleaved by signal peptid Notice, this different placement of the ’start-transfer’ sequence defines a different orientation in the membrane: N- terminus in the cytoplasm! Figure 15-17 Essential Cell Biology  Multipass membrane proteins contain multiple stop-transfer and start-transfer sequences To be clear – these sequences relate to TRANSLOCATION and NOT Translation Figure 12-49 Molecular Biology of the Cell (modified)  Many Proteins are Glycosylated on certain asparagine side-chains in the ER: ‘N-linked’ Ribosom glycosylation e not Called ‘N-linked’ shown! because the sugar chain is attached to Nitrogen on the NEVER asparagine side- occurs chain in the cytosol Functions of protein glycosylation: Helps protein folding and solubility Protects protein in harsh environments ONLY ever In some cases facilitates occurs protein sorting in the ER Can participate in lumen biological function of protein sequence in protein: ……..Asn-X-Ser/Thr…….. (where X is any amino acid excep Figure 15-24 Essential Cell Biology