Biol 4610 1st Edition Lecture 12 Outline of Current Lecture 1. Import to Mitochondrial Matrix2. Targeting of peroximal proteins3. Transport in and out of nucleus4. Molecular Mechanisms of Vesicular Budding and FusionCurrent LectureImport to the Mitochondrial Matrix- TOM: translocon of outer membrane- TIM: translocon of inner membranePath AHave N terminal matric target sequence; and an internal sequence (stop transfer sequence) à the internal sequence will become the transmembrane domainThe matrix targeting sequence means it will:1. Bind to the TOM 22/22 receptor2. Opens the TOM 40 translocon3. Opens the TIM 23 translocon4. Internal sequence (stop transfer sequence) prevents further movement through translocon1. Transmembrane domain is the stop-transfer sequencePath B proteins have a matrix targeting sequence and internal hydrophobic domains recognized by Oxa1. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Have multiple internal sequences, and they all become trans-membrane domains 1. Binds to TIM 20.22 receptor2. Opens TOM 40 translocon3. Go through TIM 23/17 transloconOXA 1 is an additional translocon. - Recognizes OXA 1 sequences and becomes the trans-membrane domain Intermembrane-space proteins have 2 pathways: Path A is predominant and proteins carry 2 different N-terminal targeting sequences. Now if we just want to get in between the two membranes1. Matrix targeting sequence binds to TOM 20/22 receptor2. Opens TOM 40 translocon3. Goes thru inner translocon TIM 23/17Intermembrane targeting sequence initially acts like a trans-membrane, but since the sequence is different it’s recognized by proteins and gets degraded by protease. So now we have the sequences cleaved off into matrix13.5. Targeting of Peroxisomal ProteinsPeroxisomal matrix proteins have a C-terminal peroxisomal targeting sequence 1 (PTS1), which has Ser-Lys-Leu at the end.Just 3 amino acids on the C terminus1. Protein is translated and folded (tertiary confirmation)2. Pex 5 receptor recognizes and binds to PTS3. Binds to and opens the PEX 10 complex (very large complex)4. Pex 5 receptor goes into the peroxisomal matrix with cargo proteinPH is much lower in peroxisomal matrix, so this lowers affinity of Pex 5 receptor and releases it à releases the cargo protein13.6. Transport into and out of the NucleusNuclear pore complex (NPC), made of nucleoporins.Big channels that are in the nucleus.F G nucleoporins (hydrophobic lining)Nuclear localization sequences (NLSs) have 7 basic aas near the C-terminus: Pro-Lys-Lys-Lys-Arg-Lys-Val. Ran: binds with GDP and then GTP, and then to ImportinImportin alpha/ beta subunits:The receptor that binds to the nuclear localization sequence.Alpha subunit binds to the NLSB subunit- on outside has hydrophobic Aas à interacts with the FG – nucleoporins that line the nuclear poreSecretory Pathway à getting proteins from the ER to the Golgi, plasma membrane, secreted into extracellular space, lysosomes- Retrograde- backward transport- Anterograde- forward transport14.2. Molecular Mechanisms of Vesicular Budding and FusionVesicles and formation of a protein coat – vesicle buds off from parents and fuses with a target membranev-SNAREs and t-SNAREs – vesicle SNARE and target snare- Recognizes where the vesicle is supposed to go to- V and t must match up with each other- All vesicles originally form bc they’re coated by a layer of proteinFormation of COPII vesiclesMove from ER to early stages in GolgiGTP binding proteins, such as Sar1, regulate coat assembly1. Receptor in ER membrane, called Sec 12 (guanine exchange factor)Pull of GDP form a protein and allows GTP to bindDiacidic sorting signal (Asp –X-Glu) - A specific AA sequence that dictates what vesicle the protein is loaded into- The purple hook is the diacidic sorting signal 2. After vesicle buds, becomes uncoated- After coat assembles, it’s releases from the ER membraneHydrolysis of GTP- Uncoats the vesicle- When sar1 goes back to GDP, tail is not in the membrane anymore- Left with uncoated vesicleRab proteins –second set of GTP binding proteins that target vesicle to appropriate membrane. - Vesicles must uncoat so vesicle SNARES can fuse with target-SNARES on golgi- When vesicle uncoats, Rab is bound to GTP- This helps to bind vesicle to new membrane- V and T snares wrap together to create SNARE complex- Dumping of vesicle cargo into the new