Protein transport and translocation 10-1 Protein transport and translocation Protein translocation in bacteria, eukaryotes targeting signals import, export systems: bacterial, ER, chloroplasts, peroxisomes, mitrochondria nuclear import

Overview of protein transport and translocation 10-2 at least 40% of all cellular proteins are: inserted into a membrane translocated into an organelle, nucleus exported outside the cell or to the periplasm proteins must be kept in translocation-competent form (i.e., either partially or entirely unfolded exception is peroxisomes, nucleus proteins must be folded/assembled after translocation; molecular chaperones are usually involved translocation is an energy dependent process

Protein translocation systems 10-3 Protein translocation systems e e i i i e IM, inner membrane IMS, inner membrane space P, periplasm OM, outer membrane TL, thylakoid lumen TM, thylakoid membrane SecYEG, Sec61, TOM, TIM, TOC are protein subunits of the translocation systems adapted from Schatz and Dobberstein, Science 271, 1519 (1996)

Targeting signals blue is hydrophilic (H-phil) 10-4 Targeting signals export signals blue is hydrophilic (H-phil) red is hydrophobic (H-phob) curling lines are helical zig-zags are turns ‘OH’ denotes hydroxylated residues ‘+’ denotes positively charged aa’s most signals are at the N-terminus can be cryptic H-phobobic H-philic H-phob H-phil H-phob import signals

Translocation in bacteria 10-5 Translocation in bacteria two major pathways for translocation in bacteria: Sec and SRP pathways both converge at SecYEG translocon and use SecA, a peripherally-bound ATPase that supplies the energy for translocation SecB binds to nascent chains containing a signal sequence and maintains the preprotein in translocation-competent form, then binds SecA; SRP docks with membrane receptor, FtsY (simpler homologues of eukaryotic SRP and SRP receptor) archaea lack SecB, have SRP/FtsY but no SecA; what drives translocation? archaeal SRP, FtsY, SecYEG more closely related to eukaryotic proteins (SecYEG)

Structure and function of SecB 10-6 SecB monomer; functional complex assembles as a tetramer (dimer of dimers) Conserved residues shown to be important for the interaction of SecB with SecA: Asp27, Glu31, Glu86 (green) Ile 84 (yellow) shading of the hydrophobic subsites 1 and 2 in the assembled tetramer; the opposite surface contains the same groove with two separate subsites 1 and 2 PTB (phosphotyrosine Binding) domain SecB monomer has an unexpected structural similarity to the PTB domain

Translocation into the ER 10-7 Translocation into the ER Sec61 is a hetero-trimeric complex composed of a, b, g subunits related to SecYEG SRP is a ribonucleoprotein complex composed of 7S RNA and numerous proteins binding of signal sequence is modulated by NAC SRP pathway is co-translational; SRP mediates arrest of elongation until it docks with SRP receptor; translocation then proceeds through Sec61 SRP is the major pathway used for import into ER a post-translational translocation pathway that makes use of Sec61 also exists; preproteins are maintained in a translocation-competent form by Hsp70/Hsp40

Folding in the Endoplasmic reticulum 10-9 Folding in the Endoplasmic reticulum

Translocation into chloroplasts 10-9 Translocation into chloroplasts Toc components, mediate translocation (Toc75 is the translocon); it is unclear how preproteins are targeted to the channel; Hsp70/Hsp40 may be involved Hsp70 in both the IMS and the stroma assist the threading of the preprotein into the chloroplast an Hsp100 chaperone also called ClpC (AAA ATPase) also binds preproteins in the stroma Hsp70/chaperonin (Cpn60) may assist folding/assembly of newly-imported protein import into thylakoids (used for respiration) uses the SRP pathway

Translocation into peroxisomes 10-10 Translocation into peroxisomes targeting of proteins is initiated post-translationally by Pex5/7 proteins, which bind the peroxisomal targeting signal (PTS) translocon not well defined; possibility of vesicular budding? gated pore that is regulated by membrane proteins? first organelle demonstrated to import proteins without a PTS, by virtue of assembly with other proteins that contained a PTS various protein oligomers are imported into peroxisomes antibodies with PTS, and 9 nm gold particles could be imported Other transport mechanisms likely involve folded proteins, including the twin-arginine (Tat) transport system of bacteria, and the cytoplasm-to-vacuole targeting pathway of yeast

Translocation into mitochondria 10-11 Translocation into mitochondria delivery of preproteins to mitochondria depends on either Hsp70/Hsp40 or MSF, mitochondrial import stimulation factor (MSF) evidence now that Hsp90 is also involved mtHsp70/Tim44/Mge (GrpE) is required for import; Tim44 contains J domain Big debate: brownian ratchet or pulling model for Hsp70 system-mediated import of proteins protein folding following import depends on Hsp70, chaperonin (Hsp60)

Import into the nucleus 10-12 Import into the nucleus nuclear localization signal (NLS) is typically highly basic; e.g., the SV40 large tumor antigen (T ag) has the sequence PKKKRKV a/b1 importin hetero-dimer recognizes and binds the NLS (or b importin alone) b importin docks with NPC and mediates interaction with Ran (GDP form) directionality conferred by nature of guanine nucleotide bound to Ran Ran binding protein (RanBP) is required for b importin binding to RanGDP; Ran GTPase activating protein (RanGAP) and nucleotide-exchange factor (RCC) are cytoplasmic and nuclear cytopl. RanGDP required for import; nuclear RanGTP required for release conversely, RanGTP binds substrate with NES in the export direction proteins to be imported can be in a native/near native form

Structure of the nuclear pore complex - RanGTP bar, 50 nm + RanGTP

Mechanism of import into nucleus 10-14 Mechanism of import into nucleus some nuclear pore proteins (nucleoporins) contain core FxFG repeats (yellow) b importin contains ‘heat’ repeats that bind the FxFG repeats (Heat repeats 5, 6, 7 are shown in red, green and blue) the FxFG repeats interdigitate in grooves formed by the Heat repeats interaction of b importin with nucleoporins allows transport across the nuclear pore complex Core FxFG repeats found in nucleoporins. Each repeat is separated by a ‘linker’ region: Bayliss et al. (2000) Cell 102, 99-108.

Heat repeat-containing protein 10-15 Heat repeat-containing protein 15 heat repeats of protein phosphatase 2A conservation is to one side of the repeat structure Groves et al. (1999) Cell 96, 99-110.