![Page 1: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/1.jpg)
Cytoplasmic Membrane Systems IILecture 12
![Page 2: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/2.jpg)
How Do Proteins Get Imported Into Membrane Enclosed Organelles?
Import Requires Input of Energy to Occur!
(Discuss with Lecture 13)
![Page 3: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/3.jpg)
The Endomembrane System Dynamic, integrated, ordered and membrane bound system
Components:Nuclear Envelope Endoplasmic ReticulumGolgi Apparatus LysosomesEndosomes Vesicles
Function in:
1)Regulating Protein Trafficking- Processing, Sorting and Transporting
2) Performing Various Metabolic Functions in the Cell
![Page 4: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/4.jpg)
The Endomembrane System
1. Transmembrane Transport:Targeting Proteins to the ER
2.Vesicular Transport: Sorting and Transport of proteins Through the Endomembrane System
![Page 5: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/5.jpg)
Components of Endomembrane System are Connected Either through Direct Contact or by Transfer of Membrane Bound Vesicles
Exocytosis
Endocytosis
![Page 6: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/6.jpg)
The Endoplasmic Reticulum is Morphologically and Functionally Subdivided
Three contiguous ER membrane domains:
Rough ER- System of Interconnected Flattened Sacs With Attached Ribosomes Proteins, lipids and carbohydrates synthesis; assembly and
folding of proteins
Transitory ER- Where vesicles exit the ER to the Golgi
Smooth ER- System of Interconnected Tubules without Ribosomes AttachedSynthesis of Lipids for Membranes: Phospholipids and
Cholesterol
![Page 7: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/7.jpg)
Functions of the Rough ER
• A major site of the synthesis of proteins, lipids and carbohydrates; assembly and folding of proteins
• Proteins produced include: membrane, secretory, lysosomal
• Best studied in cells that secrete large quantities of proteins (pancreatic enzymes; mucus-secreting cells lining the digestive tract)
![Page 8: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/8.jpg)
The Rough ER is Abundant in Cells That Secrete Large Amounts of Protein
Pancreatic Cell – secretes hormones
![Page 9: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/9.jpg)
Smooth ER Functions
• Synthesis of Lipids for Membranes: Phospholipids and Cholesterol
• Storage site for Calcium - mediated by calcium binding proteins
• Synthesis of Steroid Hormones from Cholesterol in in Specialized cell types
• In Liver Cells, Detoxifies Wide Variety of Organic Compounds (alcohol, barbiturates)
![Page 10: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/10.jpg)
The Smooth ER is Extensively Developed in Specialized Cell Types
Leydig Cell of Human Testis(Steroid Hormone Secreting Cell)
Liver Cells
Skeletal Muscle
Kidney Tubules
and Steroid Producing Glands
![Page 11: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/11.jpg)
Proteins Can Enter the Secretory Pathway by Translocation Across the ER Membrane
1.Co-Translational Targeting
Requires Recognition of a Signal Sequence by Signal Recognition Particle (SRP) in the cytosol.
Involves Translocation of Polypeptide through Gated Aqueous Channels called a Translocon
Secretory proteins or lumen resident proteins – translocated completely across the ER membrane into lumen
Transmembrane proteins are integrated into the ER membrane
2. Post-Translational Targeting
![Page 12: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/12.jpg)
Proteins use Signal Sequences for Targeting to the ER
Signal Sequence: For Soluble Proteins destined for ER Almost always N-terminal
Approximately 20 primarily hydrophobic hydrophobic
The ER Signal Sequence is Both Necessary and Sufficient to Target a Protein to the ER
![Page 13: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/13.jpg)
Cytosolic Ribosomes are Directed to the ER Membrane for Translocation of Targeted Protein
![Page 14: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/14.jpg)
Co-Translational Targeting of Protein to the ER
Involves the Signal Recognition Particle (SRP)
![Page 15: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/15.jpg)
Transmembrane Transport of A Soluble Protein into ER Lumen
2. Cleavage of Signal Sequence
By Signal Peptidase
Release of Protein Into Lumen
1. Translocon Binds Signal Sequence
Transfer of NascentProtein Across
Membrane
-Secretory Proteins-Lumen Resident Proteins
![Page 16: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/16.jpg)
Integration of a Transmembrane Protein Into the ER Membrane
Requires N-terminal ER signal sequence and a Stop Transfer Sequence
![Page 17: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/17.jpg)
Formation of a Double Pass Membrane Protein
Double Pass Membrane Protein- Internal ER signal sequence (Start Transfer Sequence)
- and a Stop Transfer Sequence
![Page 18: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/18.jpg)
Protein Folding and Processing in the ER
Lumenal Resident Proteins are involved in the folding, assembly, and modification of newly translocated polypeptides
-Protein Folding and Assembly into Multisubunit Proteins
Hsp70 Chaperone Proteins- BiPProtein Disulfide Isomerase
-Removal of the Signal Sequence
-Attachment of a Complex Carbohydrate structure (N-linked Glycosylation)
-Attachment of the phospholipid glycosylphospatidylinositol (GPI anchor)
![Page 19: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/19.jpg)
The ER Synthesizes the Major Cellular Phospholipids
Membrane Synthesis isAssociated with pre-existingmembranes
Phospholipids on cytosolic side of ER;
Glycolipids and Sphingomyelin on Lumenal surface of Golgi
Scramblase- a phospholipid translocator- ensures symmetrical growth of bilayer
Lumenal lipids become the outer leaflet of plasma membrane
![Page 20: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/20.jpg)
Lipids and Proteins Can Exit the Transitional ER by Vesicular Transport
![Page 21: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/21.jpg)
The Golgi Apparatus is a Series of Membrane Bound Compartments with Distinct Biochemical Functions
Site of Carbohydrate Synthesis
Processing, Sorting, and Dispatching of Various ER Products
Lipid Synthesis
Especially Prominent in Cells that Function In Secretion
![Page 22: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/22.jpg)
Histochemical Stains Demonstrate the Biochemical Compartmentalization of the Golgi Apparatus
Cis –osmium mannosidase II Trans-diphosphatase
![Page 23: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/23.jpg)
Protein Glycosylation
N-linked Glycosylation
(typically N X S/T Sequence)Addition occurs in the RER upon transport- quality
control checkpoint for proper protein folding
Additional modifications (trimmings and additions) occur in Golgi
O-linked – attached to OH group of Serine or Threonine Addition occurs in the Golgi
![Page 24: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/24.jpg)
Protein Glycosylation Begins in the RER
N-linked glycosylation: Addition onto an Asparagine Amino Acid
![Page 25: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/25.jpg)
Protein Glycosylation and Sorting within the Golgi
![Page 26: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/26.jpg)
Membrane-Bound Vesicles Transport Materials Through the Endomembrane System
1)Cargo Selection2) Budding3)Uncoating4)Tethering5) Docking6) Fusion 7) Recycling
![Page 27: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/27.jpg)
Membrane-Bound Vesicles Transport Materials Through the Endomembrane System
Vesicles are enclosed by proteinaceous coats
Identified coats: Clathrin, COPI and COPII
Coat functions:
1) Helps membrane bud to form vesicle
2) Cargo Selection: Captures specific components for onward transport
![Page 28: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/28.jpg)
Utilization of Different Coats in Vesicular Transport
![Page 29: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/29.jpg)
Clathrin Coated
Vesicle Formation
Clathrin binds to the membrane
Begins to assemble into a basket
Causes curvature of the membrane
(called a clathrin-coated pit)
More clathrin binds and membrane continues to curve
Eventually membrane “circle” closes
Coated vesicle pinches off
![Page 30: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/30.jpg)
Clathrin Triskelions Can Assemble
into Closed Cages
• Network of hexagons and pentagons of triskelions
• Scaffold responsible for changes in shape of the coat
![Page 31: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/31.jpg)
Vesicle Budding is Driven by Formation of Protein Coat
Clathrin- No role in Cargo selection
Adaptins- Secure Clathrin CoatSelect cargo by capturingCargo receptors
GTP hydrolysis byDynamin required For “Scission” – pinching off of vesicles
![Page 32: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/32.jpg)
Scission Requires GTP Hydrolysis by Dynamin
![Page 33: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/33.jpg)
Snares Help Direct Transport Vesicles to their Target Membranes For Fusion
Vesicles that bud carry specific marker proteins called v-SNAREs
Bind complementaryt-SNARE on target membrane
20 Different SNARES ( complementary sets) in Animal CellsEach associated with particular membrane bound organelle
![Page 34: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/34.jpg)
SNARE Pairing is Essential for Membrane Fusion
Trans SNARE
Complex
Separation of SNAREs
By NSFRequires
ATP
SNARE-SNARE Pairing provides the energy to bring two bilayers Sufficiently Close to destabilize them and result in fusion
![Page 35: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/35.jpg)
About 60 different Rabs exist- each with Characteristic Distribution
Inactive RabGDP boundCytosolic
Form
Active Rab:GTP bound Membrane
Bound
Rab Effectors- Concentrate andTether VesicleNear Target Site
Rab Contributes to Accuracy of Vesicle Targeting Before Membrane Fusion is Allowed to Occur
![Page 36: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/36.jpg)
Vesicle Trafficking is Both Anterograde and Retrograde
Why do you need Retrograde Trafficking?
![Page 37: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/37.jpg)
ER Retrieval Signals are Required to Retrieve Resident ER Proteins that Escape
Returned via Retrograde Transport
ER Membrane Proteins:KKXX Sequence- recognized by COPI Coated Transport Vesicles
ER Soluble ProteinsKDEL Sequence (Lys Asp Glu Leu)
Recognized by KDEL Receptor ( which has a KKXX Sequence)COPI Coated Vesicle Transport
Signals located at C-terminus of proteins
![Page 38: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/38.jpg)
Models for Transport Through Golgi Apparatus
Golgi Cisternae Stay in PlaceAnterograde transport of Cargoby Vesicles
Cargo Stays within a Single GolgiCisterna, which is RemodeledAnd Changes Position
Probably a Combination of Both- May depend uponCargo Size !!
![Page 39: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/39.jpg)
The Exocytic Pathway: Destination Plasma Membrane or Extracellular Space
Sorting Occurs in Trans Golgi
Specific Signal Not Known for
Targeting to Secretory Pathway
Small molecules, mucus,Digestive enzymes, hormonesAggregation of molecules To be packaged –specialSurface conditions- low pH Or high Ca2+ influences
Plasma MembraneProteins and Lipids
![Page 40: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/40.jpg)
Lysosomes Function as the the Principal Sites of Intracellular Digestion
Digests material primarily taken up by Endocytosis
Single Membrane Organelle
Has ATPase Driven H+ Pump to Maintain Acidic Lumen
Lumen Contains Many Acid Hydrolases
Lysosomal Membrane Proteins are highly Glycosylated -protects from Hydrolases in lumen
EMStained for Acid Phosphatase
![Page 41: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/41.jpg)
Proteins synthesized in ER N-linked Glycosylated
Transported to Golgi
In Cis Golgi Network Recognition of Signal Patch in Protein
Results in Addition of Multiple Mannose 6- phosphate Groups to N-glycosylation sites
Trans Golgi-
Recognition of M6P by Mannose-6-Phosphate Receptor Receptor binds Adaptins Assembly of
Clathrin Coated Vesicles
Transport to Late Endosome (M6P receptor is recycled back to Trans Golgi)
To Lysosome
Lysosomal Membrane Proteins Transport is M6P Receptor Independent
Vesicular Transport of Soluble Lysosomal Resident Proteins
![Page 42: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/42.jpg)
Endocytosis: Transport Into the Cell From the Plasma Membrane to Lysosomes
Internalize NutrientsRegulate Cell Surface Expression of ProteinsUptake and Digestion of Cellular DebrisRecovery of Membrane Inserted during Secretion
Type Based on Size of Endocytic Vesicles Formed
Pinocytosis- Ingestion of Fluid andMolecules via Small Vesicles (<200nm in diameter)
Phagocytosis- ingestion of large particles(> 200nm in diameter)
![Page 43: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/43.jpg)
Extracellular Molecules Taken up by Endocytosis are
transported to Endosomes, which mature into Lysosomes
![Page 44: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/44.jpg)
Cholesterol is Taken Up by Animal Cells by
Receptor Mediated Endocytosis
Cells outside of the liver and intestineobtain their Cholesterol from plasma rather than synthesizing de novo
![Page 45: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/45.jpg)
Receptor Uptake Involves Transcytosis in
Polarized Epithelial Cells
![Page 46: Cytoplasmic Membrane Systems II Lecture 12. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!](https://reader036.vdocument.in/reader036/viewer/2022081513/56649d585503460f94a38384/html5/thumbnails/46.jpg)
Enjoy your Spring Break!!