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What is the consistency of cytosol?

What is the consistency of cytosol?


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Imagine you somehow got a cup of pure cytosol. What would be its consistency? That is, how thick, how sticky, and how viscous would it be?


Cytosol is actually about as viscous as water (i.e. ~1 cP or 1 mPa/s).

From the Introduction of Bicknese et al. 1993

In fibroblasts and several types of epithelial cells, fluid-phase viscosity was 1.1-1.5 cP, not much higher than that of water (1 cP) (Periasamy et al., 1992). Recent measurements of cytoplasmic viscosity in CV1 and PtK1 cells by a novel ratiometric method supported the conclusion that fluid-phase viscosity in bulk cytoplasm is similar to that of water (Luby-Phelps et al., 1993).

And from the conclusion:

Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 ± 0.2 cP (fibroblast) and 1.0 ± 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane.

Interestingly, the paper cites the following studies:

In fibroblasts, fluid-phase viscosity was weakly temperature-dependent (Arrhenius activation energy 3-5 kcal/mol) and nearly independent of cell volume (Fushimi and Verkman, 1991).


Note:

cP = centipoise

  • A centipoise (cP) is a non-SI (non-System International) measurement unit of dynamic viscosity in the centimeter gram second (CGS) system of units. It is multiple of the CGS base viscosity unit named poise (P)

  • 1 cP = 0.01 g/cm/s

  • 1 cP = 1 mPa/s

Source:

Bicknese S., N. Periasamy, S. B. Shohet, and A. S. Verkman. 1993. Cytoplasmic Viscosity Near the Cell Plasma Membrane: Measurement by Evanescent Field Frequency-Domain Microfluorimetry. Biophysical Journal 65: 1272:1282


Further Reading:

  1. Beals et al 1999

  2. Gyurov & Token 2011

  3. Guthrie & Nettesheim 2012

  4. Kalwarczyk 2011


Cytosol

The cytosol, also known as cytoplasmic matrix or groundplasm, [2] is one of the liquids found inside cells (intracellular fluid (ICF)). [3] It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into many compartments.

In the eukaryotic cell, the cytosol is surrounded by the cell membrane and is part of the cytoplasm, which also comprises the mitochondria, plastids, and other organelles (but not their internal fluids and structures) the cell nucleus is separate. The cytosol is thus a liquid matrix around the organelles. In prokaryotes, most of the chemical reactions of metabolism take place in the cytosol, while a few take place in membranes or in the periplasmic space. In eukaryotes, while many metabolic pathways still occur in the cytosol, others take place within organelles.

The cytosol is a complex mixture of substances dissolved in water. Although water forms the large majority of the cytosol, its structure and properties within cells is not well understood. The concentrations of ions such as sodium and potassium in the cytosol are different to those in the extracellular fluid these differences in ion levels are important in processes such as osmoregulation, cell signaling, and the generation of action potentials in excitable cells such as endocrine, nerve and muscle cells. The cytosol also contains large amounts of macromolecules, which can alter how molecules behave, through macromolecular crowding.

Although it was once thought to be a simple solution of molecules, the cytosol has multiple levels of organization. These include concentration gradients of small molecules such as calcium, large complexes of enzymes that act together and take part in metabolic pathways, and protein complexes such as proteasomes and carboxysomes that enclose and separate parts of the cytosol.


Fun Facts about Cytoplasm

  • A different type of fluid – Cytoplasm is made up of around 80% water.
  • Cytoplasm seems to be everywhere – Cytoplasm fills up all of the spaces between the nucleus and the cell membrane. These spaces are also called “cell substance.”
  • Wait your turn – Besides letting organelles and molecules move around, cytoplasm also acts to hold them in place.
  • Ghostly references – Cytoplasm that surrounds the nucleus is called “endoplasm.” Cytoplasm close to the cell membrane is called “ectoplasm.” You might be familiar with the term “ectoplasm” as it is used in movies such as Ghost Busters, and yes, it is also gooey and jelly-like.
  • Cytoplasm by another name – There is cytoplasm inside the nucleus that is different than the cytoplasm outside the nucleus. Cytoplasm inside the nucleus is called “nucleoplasm.”
  • Pick your cytoplasm – Cytoplasm can actually be either watery or jelly-like, depending upon the activity within the cell.

Cytoskeleton

Although cytoplasm may appear to have no form or structure, it is actually highly organized. A framework of protein scaffolds called the cytoskeleton provides the cytoplasm and the cell with structure. The cytoskeleton consists of thread-like microfilaments, intermediate filaments, and microtubules that criss-cross the cytoplasm. You can see these filaments and tubules in the cells in Figure 4.5.2. As its name suggests, the cytoskeleton is like a cellular “skeleton.” It helps the cell maintain its shape and also helps to hold cell structures (like organelles) in place within the cytoplasm.


Part 2: Mechanisms of Lipid Droplet Formation

00:00:15.10 Hello, I'm Toby Walther.
00:00:17.04 And I'm Bob Farese, Jr.
00:00:19.10 And we run a joint laboratory at the Harvard School of Public Health
00:00:22.12 and Harvard Medical School.
00:00:24.05 And in this lecture,
00:00:26.16 we're gonna discuss some of the mechanisms and physiology
00:00:28.23 of lipid droplet formation.
00:00:31.14 As we mentioned in our introductory lecture,
00:00:33.28 cells have this remarkable ability
00:00:36.15 to organize the formation of lipid droplets
00:00:40.24 within the cell
00:00:42.26 in a dispersed and regular manner.
00:00:45.06 And this raises a lot of questions,
00:00:47.10 like, how do cells do this?,
00:00:48.27 what protein machinery is involved in the formation of lipid droplets?,
00:00:53.23 how do proteins target to the surfaces of these organelles?,
00:00:58.01 and what are the functions of these organelles
00:01:01.11 in cells and in organisms?
00:01:03.25 To begin with, in this lecture
00:01:07.07 we're going to show an animation made by Janet Iwasa
00:01:10.27 that illustrates a picture of how we believe lipid droplets form.
00:01:14.17 Here you can see the endoplasmic reticulum,
00:01:16.16 the site of neutral lipid synthesis,
00:01:18.29 and you see fatty acyl CoA's
00:01:21.01 and diacylglycerol substrates
00:01:23.03 that are being covalently joined
00:01:25.13 through the actions of DGAT enzymes
00:01:27.15 -- DGAT1 and DGAT2 --
00:01:29.10 to form triglycerides.
00:01:30.29 These triglycerides phase separate within the plane of the bilayer,
00:01:36.01 and accumulate via Ostwald ripening,
00:01:39.14 and then bud towards the cytosolic surface of the ER
00:01:43.25 to form a droplet.
00:01:45.17 You can see that happening here.
00:01:47.16 And as the lipid droplet grows,
00:01:49.21 the surface angle between the lipid droplet
00:01:53.14 and the ER changes,
00:01:55.14 and eventually the droplet will bud.
00:01:57.12 And the question is, how does this process happen?
00:01:59.18 And it has been our hypothesis, and others',
00:02:02.12 that proteins must be involved in the formation of these organelles.
00:02:08.02 Now, at least conceptually,
00:02:09.23 you can take apart that complex set of reactions
00:02:12.12 into individual steps.
00:02:14.07 Those don't need to necessarily occur sequentially in time,
00:02:16.25 but they can be separated for us to analyze them
00:02:19.20 one by one.
00:02:21.09 In the first step, as Bob just mentioned,
00:02:23.10 triglyceride synthesis occurs,
00:02:26.00 and the initial lens is formed.
00:02:27.25 In the next step,
00:02:30.04 we will discuss how, initially,
00:02:32.26 the lipid droplet grows and buds specifically into one direction,
00:02:35.29 towards the cytosol.
00:02:37.20 In another step,
00:02:40.21 the proteins are recruited specifically
00:02:42.29 to the surface of lipid droplets.
00:02:45.06 And when they're there,
00:02:46.21 they often catalyze reactions
00:02:49.01 that allow the expansion of the lipid droplet
00:02:50.20 in a last step.
00:02:53.14 So, let's start with step 1 of lipid droplet formation:
00:02:56.20 triglyceride synthesis and lens formation,
00:02:59.13 where triglycerides gather within the plane of the bilayer.
00:03:02.25 To begin with, in this step,
00:03:04.29 we have to go through the pathway of glycerolipid synthesis
00:03:08.10 that was discovered by Eugene Kennedy and co-workers
00:03:11.24 and reported in 1960.
00:03:14.07 And this is the major lipid synthesis pathway
00:03:16.13 that gives rise to both phospholipids
00:03:18.07 as well as the neutral lipid triglyceride.
00:03:20.20 In this pathway,
00:03:22.19 which also occurs in the endoplasmic reticulum,
00:03:24.18 fatty acids, which you can see on the far left of this slide,
00:03:28.08 are covalently linked, in a reaction requiring ATP,
00:03:31.28 by ACSL's, or acyl-CoA synthetases,
00:03:35.06 to form activated fatty acids called fatty acyl-CoA's.
00:03:39.15 These fatty acyl-CoA's, then,
00:03:42.06 are covalently linked to a glycerol backbone
00:03:44.17 in a series of enzymatic steps
00:03:46.21 to give rise to phosphatidic acid or diacylglycerol.
00:03:51.07 And those steps are catalyzed
00:03:53.05 by a series of enzymes such as GPAT enzymes
00:03:55.19 -- glycerol phosphate acyl transferases
00:03:59.25 AGPAT enzymes -- acyl glycerol phosphate acyl transferases
00:04:02.16 and the phosphate group is removed from phosphatidic acid
00:04:05.11 to form diacylglycerol by PAP,
00:04:08.13 or phosphatidic acid phosphatase.
00:04:11.11 Now, phosphatidic acid and diacylglycerol
00:04:15.04 can give rise to phospholipids,
00:04:17.17 but in the final step of making triglycerides,
00:04:20.06 diacylglycerol is covalently joined
00:04:22.21 to an acyl-CoA to form triacylglycerol
00:04:25.18 through the actions of the DGAT enzymes.
00:04:28.16 So, as I mentioned, the DGAT activity
00:04:30.27 was described around 1960,
00:04:33.24 but it wasn't for another 40 years
00:04:35.28 that we began to understand the molecular processes
00:04:38.15 of making triglycerides.
00:04:41.10 So, at the end of the 1990's,
00:04:45.11 we and folks at Calgene
00:04:49.21 identified the enzymes that make triglycerides,
00:04:51.17 and those are now known as DGAT1 and DGAT2.
00:04:54.25 So, this is fascinating.
00:04:56.16 We have two enzymes,
00:04:58.04 and these two enzymes have convergent evolution
00:05:02.09 in that they both catalyze the same biochemical step,
00:05:04.27 but they evolved separately
00:05:07.03 and are part of different gene and protein families.
00:05:09.24 So, DGAT1 is part of the MBOAT family.
00:05:12.05 DGAT2 has its own family
00:05:14.25 that constitutes DGAT's and MGAT
00:05:17.00 and wax synthases.
00:05:18.25 Both of these enzymes
00:05:20.23 are found in the endoplasmic reticulum,
00:05:22.17 but as you can see, as cartooned here,
00:05:25.02 they have very different topologies.
00:05:27.05 DGAT1 is a. is a polytopic membrane protein
00:05:30.07 with multiple transmembrane domains
00:05:32.10 and is a very hydrophobic protein.
00:05:35.06 DGAT2, on the other hand,
00:05:37.03 has one embedded transmembrane domain
00:05:39.01 that anchors it to the endoplasmic reticulum,
00:05:42.10 as diagrammed here.
00:05:46.06 Both enzymes have been the subject of
00:05:49.22 pharmaceutical inhibitor development.
00:05:51.09 And in fact, there are highly specific
00:05:53.16 and highly effective inhibitors
00:05:55.12 for both the DGAT1 and DGAT2 enzyme
00:05:57.24 that are useful both in.
00:06:00.05 for laboratory tools
00:06:04.10 and have been taken into clinical trials.
00:06:08.11 Now, currently, we only have an understanding
00:06:10.21 of the molecular mechanisms of triglyceride synthesis
00:06:13.23 catalyzed by DGAT1.
00:06:15.28 Specifically, recently,
00:06:18.18 we were able to solve the structure,
00:06:21.03 the molecular structure of this enzyme.
00:06:23.28 And as you can see here by cryo-electron microscopy,
00:06:25.29 what has become apparent is that DGAT1 generates.
00:06:29.25 is present as a dimer in the endoplasmic reticulum membrane,
00:06:34.18 and. where most of the protein
00:06:37.08 is really within the bilayer of the membrane.
00:06:40.01 It turns out that the enzyme forms, like,
00:06:43.11 a butterfly-shaped dimer
00:06:45.23 that is very intimately crossing-over
00:06:49.19 and linked between the two subunits.
00:06:52.00 When you look into one of the subunits,
00:06:53.26 it is fascinating to see how we now believe
00:06:56.14 the molecular mechanism of the triglyceride synthesis occurs.
00:06:59.25 The enzyme has a channel
00:07:02.27 that reaches from the cytosolic side
00:07:04.20 all the way through the membrane
00:07:06.12 to the luminal side.
00:07:08.27 In addition, there is a lateral cavity,
00:07:11.17 or a gate,
00:07:13.25 towards the plane of the membrane.
00:07:15.22 Deeply within the enzyme,
00:07:17.20 and deeply within the plane of the membrane,
00:07:20.02 there is the putative catalytic site.
00:07:21.24 When we look at that
00:07:25.04 in a structure that also contains substrates
00:07:27.10 and a density that most likely
00:07:29.23 is the acyl acceptor diacylglycerol,
00:07:32.21 what becomes apparent is that the acyl-CoA substrate
00:07:35.28 reaches the active site from the cytosolic side
00:07:39.11 either directly from the cytoplasm
00:07:41.10 or maybe laterally from the membrane,
00:07:43.18 and positions the carbonyl group
00:07:47.12 that is used for the esterification
00:07:50.11 right next to the acyl acceptor,
00:07:52.08 the diacylglycerol-like density,
00:07:54.16 right next to the active site.
00:07:56.21 That second substrate reaches through the lateral gate
00:08:00.20 directly to that hydrophobic core
00:08:03.00 in the middle of the enzyme.
00:08:04.24 We now think that in the next step,
00:08:06.20 the triacylglycerol is released,
00:08:08.28 possibly directly into the memory through that lateral gate,
00:08:11.18 initiating the next step,
00:08:14.05 initial lipid drop. lipid droplet growth
00:08:16.07 and lens formation.
00:08:17.29 So, as we begin to have a molecular understanding
00:08:19.26 of how triglycerides are made
00:08:21.28 and released into the membrane,
00:08:23.15 this brings us to the second step of lipid droplet formation,
00:08:25.20 and that is,
00:08:27.21 how do triglycerides phase separate
00:08:30.06 within the plane of the bilayer
00:08:31.26 and then get converted into an initial budding lipid droplet?
00:08:36.14 Now, this is a tough problem to tackle,
00:08:39.22 and we've had a lot of luck
00:08:41.23 taking systems-type approaches
00:08:43.09 to identify the machinery of lipid droplet formation.
00:08:45.22 What's shown here is a screen
00:08:47.24 that we recently performed
00:08:49.18 in human macrophage cells
00:08:51.06 that were loaded with both cholesterol and triglycerides
00:08:54.04 to form lipid droplets,
00:08:55.12 and then we used RNAi
00:08:58.02 to knock down the genes in the genome
00:09:00.06 and determine which of these genes
00:09:02.12 was required for normal droplet formation.
00:09:04.20 And from a screen like this,
00:09:06.10 we obtained, as you can see,
00:09:08.08 500 hits for. that are involved in lipid droplet biology.
00:09:11.28 Now, from image analysis,
00:09:14.22 we can classify the characteristics
00:09:17.11 of how these different gene inhibitions
00:09:21.07 affect lipid droplet biology,
00:09:23.18 and then bin them out into different phenotypes, as you can see.
00:09:26.21 So, we have some classes
00:09:29.10 where there are very small and dispersed droplets,
00:09:31.23 and other classes where the droplets are quite large and clustered,
00:09:34.05 for example.
00:09:36.06 Some examples of these different classes
00:09:38.06 can be seen in these images,
00:09:40.01 where, again,
00:09:41.26 we have a variety of very strong lipid droplet phenotypes
00:09:44.01 caused by individual gene knockdowns.
00:09:46.20 Some of the genes that cause these different phenotypes
00:09:49.25 are shown on the right, here,
00:09:51.21 and obviously, today we can't discuss
00:09:54.01 many of the hits from the screen.
00:09:55.15 I will say, though,
00:09:57.22 all these hits will be made publicly available
00:09:59.16 on a lipid droplet platform
00:10:01.25 that will be launched very soon.
00:10:04.09 One of the hits that we would like to talk about today
00:10:06.25 is this one called BSCL2 or seipin.
00:10:10.04 BSCL2 is the gene that encodes for the seipin protein,
00:10:12.28 and this hit was one of the strongest hits in our screen,
00:10:16.06 and of course attracted our interest
00:10:18.00 because we and others
00:10:21.22 have identified seipin as a very important protein
00:10:24.17 in lipid droplet formation.
00:10:26.19 So, seipin. what is seipin?
00:10:28.13 seipin is an ER protein,
00:10:30.20 as shown in the cartoon on your left,
00:10:32.22 which has N- and C-termini
00:10:34.14 that are oriented towards the cytosol.
00:10:36.18 It has two transmembrane domains
00:10:39.02 and an evolutionarily conserved loop
00:10:42.15 that is oriented towards the lumen of the ER.
00:10:45.26 And seipin was identified in 2001
00:10:48.18 as the causative gene.
00:10:52.00 one of the causative genes for congenital generalized lipodystrophy,
00:10:56.15 which. an example of that syndrome,
00:10:59.07 where there is a lack of body fat,
00:11:01.19 is shown in the image on your right.
00:11:03.09 Now, over the years,
00:11:05.17 we and many other labs have worked on the seipin protein
00:11:08.05 because many labs have identified it
00:11:10.25 as being central to lipid droplet formation,
00:11:13.03 and some of those labs are listed on the bottom.
00:11:16.15 Some of the major findings
00:11:19.10 that are important for understanding
00:11:22.07 seipin's potential role in lipid droplet formation
00:11:24.13 are listed on this slide.
00:11:25.20 One is that the deletion of seipin
00:11:26.28 -- which was first identified as.
00:11:29.09 done in screens in yeast by the Goodman Lab
00:11:32.08 or Rob Yang's lab --
00:11:33.16 show that seipin deletion
00:11:35.25 results in supersized lipid droplets,
00:11:37.00 very giant lipid droplets found in the cells.
00:11:39.23 Joel Goodman's lab also originally showed
00:11:42.01 that seipin homo-oligomerizes
00:11:44.06 to form a large macromolecular complex.
00:11:47.00 And a number of labs also showed
00:11:50.26 that seipin localizes to ER-lipid droplet junctions in yeast.
00:11:55.26 Now, this slide shows a figure in which
00:11:59.06 we're showing you the cellular phenotype
00:12:01.08 for seipin deletion.
00:12:02.28 On your left, you can see that
00:12:04.29 lipid droplets form in these Drosophila cells
00:12:06.18 in an organized manner,
00:12:08.28 as we showed you in some of the previous videos.
00:12:11.10 We load with oleate, they form triglycerides,
00:12:13.05 and droplets form in a dispersed manner.
00:12:16.06 On your right is what happens
00:12:18.01 in seipin deficient cells.
00:12:20.05 And in this case, what we see is something completely different.
00:12:24.01 There are some pre-existing large lipid droplets,
00:12:25.29 but instead of having normal-sized lipid droplets,
00:12:30.05 what we see is a myriad of very small lipid droplets
00:12:32.27 that are found throughout the endoplasmic reticulum,
00:12:37.01 that look distinctly different than the normal-sized droplets
00:12:41.26 found in the wild-type situation.
00:12:43.29 Now, how does the protein actually do this?
00:12:46.02 An interesting early observation in our laboratory
00:12:49.02 was that when we actually look.
00:12:51.19 how the protein looks like,
00:12:53.19 it forms foci in the endoplasmic reticulum.
00:12:56.18 What you can see in the movie on the left
00:12:58.25 is a Drosophila cell, just like the one Bob just showed,
00:13:01.16 where we've genome-engineered the cells
00:13:04.26 to express a version of seipin
00:13:06.20 that has a GFP right at the end of a protein,
00:13:09.01 at its endogenous locus.
00:13:11.06 And what you can see is that this cell now
00:13:14.10 has these green foci,
00:13:16.02 which move rapidly through the endoplasmic reticulum
00:13:18.22 -- now shown in red here --
00:13:20.12 almost as if they were just gonna scan the ER
00:13:23.04 for initial lipid droplet formation.
00:13:25.27 And it turns out that this is a conserved feature
00:13:28.24 for seipin,
00:13:30.13 because it's not only forming punctae in Drosophila cells,
00:13:33.02 but as you can see on the right, here,
00:13:35.17 also in mammalian cells,
00:13:37.01 and the same is true in many other systems
00:13:38.19 where people have looked.
00:13:40.13 Now, what's the molecular nature of these foci?
00:13:42.26 Our current understanding
00:13:45.02 is driven by observations,
00:13:47.02 again, by cryo-electron microscopy,
00:13:49.04 where we have isolated and purified seipin,
00:13:52.27 in this case, from Drosophila cells.
00:13:54.28 And what you observe is that
00:13:58.04 seipin has sort of three parts to it.
00:14:00.13 There are two short extensions on the N- and the C-terminal part
00:14:04.22 that are both predicted to face the cytosolic side.
00:14:08.03 Each subunit also has two transmembrane domains
00:14:10.09 that span the ER membrane.
00:14:13.13 And then there is a large,
00:14:15.09 quite conserved luminal domain,
00:14:16.26 now shown in green in the cartoon.
00:14:20.18 In our cryo-EM structure, what we observe is
00:14:27.00 particularly that the luminal domain folds
00:14:29.27 into a very, very solid alpha/beta fold,
00:14:32.00 that resembles lipid-binding proteins,
00:14:34.07 that arrange into a ring structure
00:14:39.17 that contains 10, 11, or 12 subunits,
00:14:41.08 depending on the species.
00:14:42.29 You can also see that the transmembrane domains
00:14:45.18 are above that
00:14:47.11 and span the bilayer membrane,
00:14:49.17 but in the structure,
00:14:51.01 those were not resolved,
00:14:52.17 presumably because they were quite flexible.
00:14:55.16 Now, analyzing the structure of the folded domain
00:14:58.08 in the luminal side,
00:14:59.23 what we observed is two particular features.
00:15:03.12 One is that this ring really underlays
00:15:06.18 right under the membrane,
00:15:08.03 resembling what could be called a molecular washer
00:15:09.24 that basically anchors the whole machinery
00:15:14.00 -- you know, a very large protein complex at this point,
00:15:16.18 with, like, 12 subunits --
00:15:18.06 right under the ER.
00:15:20.02 The second feature that seems to be specific for seipin
00:15:22.20 in this class of related molecules
00:15:25.25 is that there is a helix
00:15:29.16 that is present in all seipin molecules
00:15:32.06 that have been described today
00:15:34.10 that is pressed and oriented right onto the membrane.
00:15:38.06 This hydrophobic helix, in isolation,
00:15:40.06 suffices to bind triglyceride,
00:15:42.14 leading us to the model that this complex
00:15:45.15 organizes the original.
00:15:47.04 the initial formation of lipid droplets.
00:15:50.05 Having said that, this model predicts, of course,
00:15:55.06 that one can isolate triglyceride in this complex
00:15:57.01 as it is formed.
00:15:58.21 And we've tried that and never succeeded with it.
00:16:01.03 There's a second puzzle here,
00:16:03.04 and that is that this hydrophobic helix
00:16:05.21 is highly evolutionarily conserved in sequence,
00:16:08.12 a feature that you would not necessarily expect
00:16:10.17 if the only role of this was to be hydrophobic
00:16:13.02 and interact with oil.
00:16:14.29 So, based on this, we had a puzzle,
00:16:18.02 and we hypothesized there may be
00:16:20.26 a missing component to this assembly complex.
00:16:22.20 And here is an experiment
00:16:24.28 that Jeeyun Chung in our lab carried out,
00:16:26.26 in which she did an immunoprecipitation,
00:16:29.06 or a pulldown experiment,
00:16:30.28 using either the wild type seipin,
00:16:33.02 which is shown on your left,
00:16:34.27 or a seipin molecule
00:16:37.18 in which she deleted this highly conserved hydrophobic helix,
00:16:39.19 which is shown on the right.
00:16:41.05 And in pulling down all the interactors,
00:16:43.05 she found one hit
00:16:48.01 that was pulled down specifically with the wild type protein
00:16:49.21 but not with the mutant protein that deleted this hydrophobic helix.
00:16:52.06 And that is very easy to see
00:16:55.11 in the upper right part of the figure, here,
00:16:57.15 and that gen.
00:17:00.06 the protein that was pulled down by the wild type
00:17:01.22 is a protein that was named TMEM159.
00:17:04.25 So, what is TMEM159?
00:17:07.01 Well, so.
00:17:12.03 we have now named TMEM159 lipid droplet assembly factor,
00:17:14.16 and so that name speaks to what we think.
00:17:17.27 its involvement in lipid droplet assembly.
00:17:21.03 So, not much was known about this
00:17:24.01 when we identified it as a seipin interactor.
00:17:25.22 TMEM159, or LDAF1,
00:17:27.17 is an endoplasmic reticulum protein.
00:17:29.27 161 amino acids.
00:17:32.14 Its topology suggests both the N- and C-termini
00:17:36.01 are towards the cytoplasm.
00:17:37.21 And the characteristics of the protein
00:17:40.17 suggest that it forms a double hairpin,
00:17:45.02 as we've modelled in this cartoon.
00:17:47.22 The gene and protein were originally identified
00:17:49.23 in murine fatty liver
00:17:51.10 that had been induced by PPAR-gamma
00:17:53.00 in a knockout model
00:17:55.07 where there was fatty liver.
00:17:57.23 And one of the things that caught our attention
00:18:00.00 when we did this pulldown
00:18:01.04 was it turned out that the knockdown
00:18:04.24 of TMEM159 or LDAF1
00:18:06.28 also was highly correlated with seipin
00:18:09.16 in the genome-wide screen that I showed you previously.
00:18:12.22 So, Jeeyun went on to purify LDAF1,
00:18:17.07 as I'll refer to it now,
00:18:20.08 as shown here, from mammalian cells.
00:18:21.22 And when she pulls down and purifies LDAF1,
00:18:23.22 she also copurifies seipin.
00:18:28.15 And these two proteins
00:18:30.19 now form a stoichiometric complex.
00:18:32.24 Importantly, as Toby mentioned before,
00:18:34.24 when we purified seipin alone before,
00:18:37.18 we were unable to copurify triacylglycerol with seipin.
00:18:41.15 But we made the discovery here
00:18:44.20 that when we purify the complex of both proteins together,
00:18:47.16 as you can see in the fourth from the left lane,
00:18:52.20 the complex pulled down in the Coomassie gel on the bottom
00:18:55.08 also shows the presence of triacylglycerol.
00:18:57.14 If we add oleic acid to the cells
00:18:59.20 and drive lipid droplet formation,
00:19:01.11 the complex purifies even more triacylglycerol,
00:19:04.00 as you can see.
00:19:05.19 And if we used DGAT inhibitors
00:19:07.24 in that situation,
00:19:09.09 we blocked the triacylglycerol.
00:19:10.29 So, this is all evidence to us that this complex,
00:19:13.05 together,
00:19:15.06 is interacting with triacylglycerol
00:19:17.23 during lipid droplet formation.
00:19:20.14 We went on to study LDAF1
00:19:23.27 in the context of cells,
00:19:25.22 and one of those experiments is shown here.
00:19:28.08 In this experiment,
00:19:29.26 what has been done is that
00:19:32.09 LDAF1 and seipin have been tagged,
00:19:34.16 using CRISPR technology,
00:19:36.04 at their endogenous loci with fluorescent proteins.
00:19:40.04 In addition, we tagged PLIN3,
00:19:42.23 which we have identified as one of
00:19:45.25 the earliest detectors of a forming lipid droplet.
00:19:48.00 And so, shown in this slide
00:19:49.28 are three examples of lipid droplets forming,
00:19:52.12 as detected by PLIN3 signal,
00:19:55.17 on the bottom set of.
00:19:58.27 in the bottom row of these panels.
00:20:00.15 And in all instances,
00:20:01.16 when we can first detect the lipid droplet formation,
00:20:04.11 it is occurring in the same spot
00:20:06.27 as where we have LDAF1 and seipin.
00:20:08.17 So, this gives us
00:20:11.11 more evidence that the seipin-LDAF1 complex
00:20:14.00 is the site where lipid droplets
00:20:16.27 are forming in these cells.
00:20:19.15 Now, if this model is correct,
00:20:21.14 there are a couple of simple predictions.
00:20:23.18 One of the strongest predictions is that,
00:20:26.06 if the complex determines where lipid droplets form,
00:20:28.27 we should be able to move the complex
00:20:31.11 to a site where it normally is not localized,
00:20:33.10 or where only a few of them are randomly,
00:20:35.10 and now lipid droplets should form,
00:20:37.27 preferably, at those sites.
00:20:39.29 In this experiment, we used a chemical biology trick
00:20:42.21 in which we've generated
00:20:45.07 an artificial chemically induced heterodimerizing version
00:20:49.12 of LDAF1
00:20:51.20 that can be bound to a plasma membrane anchor.
00:20:54.20 When we add to cells that express those two constructs
00:20:59.06 a chemical heterodimerizer,
00:21:00.20 what occurs is that those two parts of the proteins
00:21:03.20 form a complex,
00:21:05.19 and that drags the whole endoplasmic reticulum
00:21:08.04 in close proximity to the plasma membrane.
00:21:11.19 Normally, there is only very little plasma.
00:21:13.29 endoplasmic reticulum right under the plasma membrane.
00:21:16.20 But when we induce this,
00:21:18.23 what we observe is.
00:21:20.07 by total internal reflection microscopy.
00:21:22.19 is that both LDAF1 is targeted,
00:21:25.13 as you would predict,
00:21:27.01 but also seipin is now brought along
00:21:28.18 because it is in a protein-protein complex with LDAF1,
00:21:31.21 not directly interacting with the anchor.
00:21:35.03 This now generates a scenario
00:21:37.26 in which right underneath the plasma membrane
00:21:40.00 we have a lot, a lot of ER
00:21:42.15 that's tethered to it.
00:21:44.05 And what's important and interesting is
00:21:46.27 when we now look at lipid droplet formation,
00:21:49.01 we see that now lots of, lots of lipid droplets form
00:21:51.24 underneath the plasma membrane,
00:21:53.20 because now you have this complex there,
00:21:55.12 which normally isn't found there.
00:21:57.04 In a wild type cell that doesn't have.
00:21:59.01 or in a cell that hasn't been exposed to the dimerizer,
00:22:01.16 a control cell,
00:22:03.02 we see only very, very few droplets
00:22:05.03 forming right under the plasma membrane.
00:22:07.22 But as you can see in the quantitation on the right, here,
00:22:11.07 or in the stills taken from a movie on the left,
00:22:13.03 as soon as you bring this complex right underneath the plasma membrane,
00:22:16.03 now, lots of, lots of lipid droplets form there,
00:22:18.18 because now the machinery is there
00:22:20.04 to catalyze this reaction.
00:22:23.15 So, these are some of the highlights
00:22:25.16 of studies where we have identified
00:22:28.01 at least a couple components
00:22:30.07 of a lipid droplet assembly complex:
00:22:32.17 seipin, which we and others
00:22:34.24 had identified over the years
00:22:36.25 as being crucial to this complex
00:22:38.17 and this other protein, LDAF1.
00:22:40.10 And what's shown here is our working model
00:22:42.13 for how this might work.
00:22:44.03 So, as we mentioned,
00:22:46.02 triglycerides are made through the actions of, for example,
00:22:48.22 DGAT enzymes in the endoplasmic reticulum.
00:22:50.14 And they are initially released into the plane of the bilayer.
00:22:55.06 It's our hypothesis, then,
00:22:56.29 at the moment,
00:22:58.23 that the seipin-LDAF1 complex
00:23:01.06 becomes the site
00:23:03.28 where these triglycerides can nucleate and phase separate
00:23:06.00 to begin to form lipid droplets.
00:23:07.25 And what's really intriguing
00:23:09.23 is this complex brings together, for example, in humans,
00:23:12.19 66 membrane-spanning domains in a very small area
00:23:16.18 that might create hydrophobic surfaces
00:23:19.04 for this. for this nucleation.
00:23:21.26 catalytic step to occur.
00:23:23.24 So, shown here, then,
00:23:25.29 are triglyceride molecules
00:23:28.07 that have begun to form a lens
00:23:30.07 in the setting of this complex.
00:23:31.29 And as you can see, LDAF1 begins to
00:23:36.14 provide curvature to the membrane,
00:23:38.02 which may also facilitate bending of the membrane
00:23:40.09 and droplet formation.
00:23:42.27 The droplet then grows.
00:23:44.26 And what we didn't show you,
00:23:46.11 but we have data indicating,
00:23:48.02 is that as the droplet is growing
00:23:50.15 LDAF1 coats the surface of the droplet.
00:23:52.12 Here, we think LDAF1 provides surface-modulating qualities,
00:23:56.18 such as lowering the surface tension
00:23:58.06 and regulating some of the protein composition
00:24:00.03 of the developing droplet.
00:24:02.13 So, that's the current model.
00:24:04.01 And obviously, this model will require further testing.
00:24:06.26 And one other aspect of this model
00:24:09.14 is that, as Toby mentioned before,
00:24:11.16 there are domains of seipin
00:24:13.25 that appear very similar
00:24:16.20 to lipid-binding domains,
00:24:18.04 so it may be that this complex
00:24:20.12 also has some role in directing lipid trafficking
00:24:22.21 -- whether that be neutral lipids or phospholipids --
00:24:25.22 during the formation of lipid droplets.
00:24:28.02 That also remains to be further tested.
00:24:31.11 So, in this part of our lectures,
00:24:32.24 we told you about triglyceride synthesis
00:24:34.20 and the initial stages of lipid droplet formation
00:24:37.02 at the endoplasmic reticulum.
00:24:38.24 But what happens next?
00:24:40.11 How do the lipid droplets acquire their specific proteome?
00:24:43.13 How do they grow in size
00:24:45.15 and detach from the ER?
00:24:46.26 And what are their functions in the cell?
00:24:49.09 So, we'll tell you about that in the next part of our lectures.
00:24:52.11 Stay tuned.


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142 Cards in this Set

The capturing and using of the energy of living systems.

How do cells get the energy they need?

Chemical sources (respiration) or the sun (photosynthesis)

What are the primary energy sources that the cell can use?

Complex carbs have storage properties. What are the two storage forms of glucose?

Glycogen (animal cells) and Starch (plant cells)

What component of fat is the highest source of energy?

When sugars and fats are broken down, their energy must be captured and stored in forms that can be readily used called:

Give some examples of activated carriers.

ATP, Acetyl CoA, NADH, NADPH, FADH2

What percentage of cellular energy is used to make proteins?

The energy available to do work is called:

Free energy or G (Gibbs Free Energy)

For a chemical reaction, if a reactant/substrate X has a greater energy than product Y:

For a chemical reaction, if a product Y has a greater energy than reactant/substrate X:

If ΔG is negative, the reaction is:

If ΔG is positive, the reaction is:

If ΔG is 0, the reaction is:

The breakdown of glucose makes a lot of energy. The reaction:

Glucose + 6 O2 --> 6CO2 + 6 H2O = ___ kcal/Mol

Rank the energy content of the following: ATP, ADP, AMP

What are the three mechanisms to generate ATP?

Photophosphorylation, Oxidative Phosphorylation, and Substrate Level Phosphorylation

This ATP generating mechanism involves the absorption of sunlight coupled to ATP synthesis.

This ATP generating mechanism involves energy from activated carriers (NADH, FADH2) coupled with ATP synthesis (paired with the electron transport chain)

This ATP generating mechanism uses enzymes to catalyze reactions coupled directly to ATP synthesis.

Substrate Level Phosphorylation

When O2 is available, what percentage of energy may be captured and stored as ATP?

Where does the Krebs cycle occur?

Describe the overview of Glycolysis.

-Occurs in both the presence and absence of oxygen

Objective: To make ATP from energy extracted from sugars.

What are the products of glycolysis?

Glucose is broken down into 2 pyruvate molecules through glycolysis. From here, it can take one of three pathways. Name them and indicate their oxygen requirements.

>Krebs Cycle (requires oxygen)

>Lactic Acid (does not requires oxygen)

>Ethanol (does not require oxygen)

What determines the path that the pyruvates will take post-glycolysis?

The process by which 2 pyruvates are turned into lactic acid is also called:

Describe the process of pyruvate being converted into lactic acid.

>No additional ATP is made

>There is energy in lactate, but under these conditions, the cell cannot extract this energy.

2 pyruvates, under anaerobic conditions, can turn into Lactate or ____ + waste ____

Where does Lactic Acid and Ethanol + CO2 production coupled with glycolysis occur?

In the cytosol in the absence of oxygen

True or False: Fermentation and Ethanol production both produce additional ATP when paired with glycolysis which produces 2 ATP.

FALSE there is a net production of 2 ATP only from glycolysis. No extra ATP is produced from fermentation or ethanol production.

Anaerobic Respiration captures ___ % of energy from glucose.

Aerobic Respiration captures ___% of oxygen from glucose.

In the presence of Oxygen, Pyruvate is imported into the mitochondria and oxidized into this compound, which can enter the Krebs Cycle.

TRUE OR FALSE: Though often drawn together, the Krebs Cycle and the oxidation of pyruvate are actually two separate processes.

The oxidation of pyruvate (pyruvate --> Acetyl CoA) is actually three reactions catalyzed by three enzymes at the same place. This three enzyme complex is called:

Pyruvate Dehydrogenase Complex

How many reactions occur in the Krebs Cycle that extract energy from Acetyl CoA?

Describe the products of the Krebs Cycle

2 Pyruvates --> 2 Acetyl CoA -->

>2 ATP (used to do cellular work)

>8 NADH (high energy transfer electrons in ETC)

In what reactions is NADH produced? FADH2?

NADH - Glycolysis, Krebs Cycle, Oxidation of Pyruvate

What kind of fats are most often used as an energy source?

Triglycerides (3 fatty acid tails)

Where does the β-Oxidation of Fatty Acids occur?

What happens during the β-oxidation of fatty acids?

-Fats and Lipids are broken down by enzymes into fatty acids

-These acids are oxidized into Acetyl CoA

-FADH2 is produced, NADH is produced, and Acetyl CoA is produced which can enter the Krebs Cycle

-Fatty acid tails have a lot of carbon atoms, so more energy can be produced

-FADH2 and NADH donate electrons in the electron transport chain

β-Oxidation can also occur in this region, but no ATP is used.

Describe what happens during β-oxidation in the peroxisome.

>Acetyl CoA is exported to the cytosol

>NADH is exported to the cytosol

>FADH2 is used to break down hydrogen peroxide

What is the objective of the peroxisome?

This part of the mitochondria surrounds the organelle completely and has big porins in it that allow rapid traffic to pass. It is selectively permeable, but a lot of things are allowed to cross because the porins are so big.

In this area of the mitochondria, many reactions occur.

This portion of the mitochondria runs along the outer membrane and occasionally extends finger-like projections into the outer membrane. By folding, it greatly increases surface area. Two things that happen here: Electron Transport reactions and ATP synthesis

This is the aqueous region inside the inner membrane that has the consistency of cytosol. Certain reactions occur here.

What percentage of the organism's genome does mitochondrial DNA make up? (plants and animals)

Where are most proteins in the mitochondria made?

They are nuclear encoded and synthesized in the cytosol, then transported into the mitochondria

List processes that occur within the mitochondria.

>Citric Acid/Krebs Cycle (matrix)

>Beta Oxidation of fatty acids

>Electron transport chain (inner membrane)

>ATP synthesis (Inner membrane)

Describe the electron transport chain's composition.

A chain/group of large protein complexes and some smaller proteins that are embedded in the membrane. They accept and donate electrons from FADH2 and NADH. This is a series of REDOX reactions that move substances through the complexes.

NADH has to enter the electron transport chain (moved from cytosol into mitochondria) but it is NOT permeable to the membrane. How does it enter?

How does the malate shuttle work?

The energetic equivalent of NADH is moved across the inner membrane because NADH cannot get across.

Oxaloacetate picks up the high energy electrons from NADH and gives them to something else that can carry them across. Oxaloacetate is converted to malate which CAN move across, and when it is across the membrane, it reclaims its electrons.

This mechanism moves pyruvate from the cytosol into the mitochondrial matrix.

True or False: The glycolysis products (NADH and pyruvate) are moved into the mitochondria with a proton gradient and malate shuttle. The products of pyruvate oxidation and the citric acid cycle (NADH, FADH2, ATP, Acetyl CoA) are in the mitochondria, but in the wrong part.

This is a protein complex that uses the energy of a proton gradient to make ATP. All the energy from FADH2 and NADH is converted to ATP through this. It is located in the inner membrane.

True or False: The energy used to move protons from one side of the electron transport chain to the other is used to generate a pH gradient.

True or False: NADH and FADH2 donate their electrons at the same location in the electron transport chain.

What is the role of Oxygen in the electron transport chain?

Oxygen is the terminal electron acceptor (at the end of the chain). Electrons must be pulled out of the chain. As electrons are donated and passed through complexes, water is formed and holds onto some electrons.

What is the reaction corresponding to oxygen's purpose in the electron transport chain?

Which protein allows oxygen to act as the terminal electron acceptor?

Cytochrome oxidase complex

True or False: The electron transport chain converts one type of active carrier into another.

True NADH or FADH2 into ATP

The ATP synthase is coupled to the proton gradient. How do these two work together?

1. The energy of the electron transport chain is used to pump protons across the membrane.

2. The energy in the proton gradient is harnessed by ATP synthase to make ATP

What is the coupling of the proton gradient and synthesis of ATP called?

Where does chemiosmosis occur and what type of phosphorylation occurs in each area?

Mitochondria (oxidative phosphorylation)

Chloroplasts/thylakoid membrane (photophosphorylation)

This concept is described as the electron flow through the electron transport chain pumps protons across the membrane.

How are proton gradients used in bacterial cells?

There are certain molecules that, when embedded in the membrane, they collapse the ion gradients. It's like poking holes into the membrane, and the energy source is suddenly lost. Molecules that do this are called:

How much ATP is generated by aerobic metabolism for one molecule of glucose per product?

-Some ATP is produced directly by substrate level phosphorylation (Glycolysis?)

What are the products of glycolysis, the oxidation of pyruvate, and the krebs cycle? How much ATP do they produce?

TOTAL = 36 ATP (theoretical yield)

What is the theoretical yield and observed yield of aerobic respiration?

What are some causes of lower ATP yield in aerobic respiration?

Inner mitochondrial membranes leak protons and proton gradient is used for other purposes

Compare efficiency in aerobic and anaerobic respiration

We break down glucose, carbons, hydrogens and oxygens end up in water, and the energy associated with all chemical bonds is captured in ATP. What happens to the rest of the energy?

What do we call organisms that can retain the heat produced by ATP production?

This is the outermost structure of the plant cell, residing outside of the cell membrane. It is made of cellulose and other types of polysaccharides. It's very complex and does not break down well.

This structure of the plant cell is found in mature cells and is surrounded by a membrane. It can sometimes be so big that it can push all other organelles against the cell membrane. It can occupy 80-95% of the cellular space. It contains water, and the hydrostatic pressure is what gives plant cells their support.

This is a group of double-membrane enclosed organelles.

Amyloplasts and Chloroplasts

These plastids hold starch and sometimes can hold so much starch that they look like they are empty. They are used by plant cells to detect gravity and are similar to statoliths in the inner ear.

These plastids are found in plants and algae. Their numbers vary from 20-50 per cell. Some bacteria are photosynthetic but do not contain these.

How are mitochondria similar to chloroplasts?

They both have double membranes (envelope)

They both contain their own DNA

They can make proteins, but not a lot of proteins. Most are made in the cytosol and are imported into the organelle.

This chloroplast component is the part that is not occupied by the membranes. It is aqueous based.

The stroma is similar to what component of the mitochondria?

This component of the chloroplast is sometimes stacked up like pancakes, but sometimes they are individual in the cell.

If we took the thylakoid membrane and sliced them open, we see that they have spaces inside. These spaces, cavities, or openings are called:

Thylakoid Lumen/Thylakoid spaces

What percentage of cellular DNA is the DNA in chloroplasts?

In what three places can we find DNA in cells

Mitochondria, chloroplasts, nucleus

In what three places can proteins be synthesized?

In cytosol, in mitochondria, in chloroplasts

What are the three objectives in photosynthesis?

a.) Capture solar energy and convert it into chemical energy for the use of other living things

b.) Can use inorganic carbons (CO2) and convert them to organic carbon (sugars)

c.) Source of atmospheric oxygen (O2) released as waste

What is the summary equation of photosynthesis?

6 CO2 + 6 H2O --> 6 (CH2O) + 6 O2

What does each component in oxygenic photosynthesis do?

H2O -> Used as a source of electrons and hydrogens

O2 - Released from the water molecule

The type of photosynthesis that releases oxygen, particularly by plants, algae and cyanobacteria is called

What is the alternative form of photosynthesis called and what is its equation?

What does each component in anoxygenic photosynthesis do?

H2S - Source of protons and electrons (rotten egg smell)

Where do light reactions occur?

Where do dark reactions occur?

What occurs during the light reactions?

Light energy is absorbed and converted to chemical energy (ATP, NADH) using various pigments

Chlorophyll and other pigments function to absorb light energy in a structure/unit called a:

What are the three components of a photosystem?

-Chlorophyll acting as an antennae

-Chlorophyll acting as a reaction center

What occurs in the reaction center in the photosystem?

Light energy in the chlorophyll molecules excite the electrons, bringing them to a higher energy levelP

Photosystems are part of the

Photosystem II can break apart water molecules and extract the electrons and proteins from this water. The electrons are used to replace the electrons lost in the reaction center chlorophyll. This is called

What happens to the products of photolysis

Electrons are donated to chlorophyll to replace lost electrons

Hydrogen accumulates in the thylakoid lumen (later will made a proton gradient)

Oxygen is released in the atmosphere

ATP synthesis is this type of phosphorylation.

Photophosphorylation is a type of

What are the three major pathways involved in dark reactions?

Describe the process of the C3 pathway

First reaction: CO2 + RuBP --> 2 PGA

PGA is further metabolized by the calvin cycle which requires a lot of ATP and NADPH to produce G3P

Rubisco catalyzes two reactions. What are they?

CO2 + RuBP --> PGA is the first step in what process?

O2 + RuBP --> PGA + PGLY is the first step in what process?

The photorespiration pathway metabolizes what?

The photorespiration pathway occurs in three organelles. What are they?

Chloroplast, peroxisome, mitochondria

This is the idea what photorespiration results in the loss of CO2 and therefore lowers the rate of photosynthesis.

What determines whether rubisco uses CO2 or O2?

The relative amount closest to Rubisco

C4 and CAM attempt to shove carbon dioxide closer to rubisco and prevent photosynthesis from being reduced. C4 is the basic carbon fixation pathway (Calvin Cycle). C4 and CAM modify the C3 pathway, elevating CO2 at rubisco. This reduces photorespiration and increases photosynthesis.

This is the degradative process by which large molecules are broken down into smaller ones. Usually energy is released, starting with high energy through a step by step process. Energy is captured and stored in activated carriers.

This is a biosynthetic pathway used to build up substances. It requires energy.


Extracellular Fluid Composition

The extracellular fluid is mainly cations and anions. The cations include: sodium (Na+ = 136-145 mEq/L), potassium (K+ = 3.5-5.5 mEq/L) and calcium (Ca2+ = 8.4-10.5 mEq/L). Anions include: chloride ( mEq/L) and hydrogen carbonate (HCO3- 22-26 mM). These ions are important for water transport throughout the body.

Plasma is mostly water (93% by volume) and contains dissolved proteins (the major proteins are fibrinogens, globulins, and albumins), glucose, clotting factors, mineral ions (Na+, Ca++, Mg++, HCO3- Cl- etc.), hormones and carbon dioxide (plasma being the main medium for excretory product transportation). These dissolved substances are involved in many varied physiological processes, such as gas exchange, immune system function, and drug distribution throughout the body.


Cellular Dimensions

Most cells are of microscopic size. Animal and plant cells are typically 10 to 30 μm in diameter, and many bacteria are only 1 to 2 μm long.

What limits the dimensions of a cell? The lower limit is probably set by the minimum number of each of the different biomolecules required by the cell. The smallest complete cells, certain bacteria known collectively as mycoplasma, are 300 nm in diameter and have a volume of about 10 -14 mL. A single ribosome is about 20 nm in its longest dimension, so a few ribosomes take up a substantial fraction of the cell's volume. In a cell of this size, a 1 μM solution of a compound represents only 6,000 molecules.

Figure 2-2 Smaller cells have larger ratios of surface area to volume, and their interiors are therefore more accessible to substances diffusing into the cell through the surface. When the large cube (representing a large cell) is subdivided into many smaller cubes (cells), the total surface area increases greatly without a change in the total volume, and the surface-to-volume ratio increases accordingly.

The upper limit of cell size is set by the rate of difl'usion of solute molecules in aqueous systems. The availability of fuels and essential nutrients from the surrounding medium is sometimes limited by the rate of their diffusion to all regions of the cell. A bacterial cell that depends upon oxygen-consuming reactions for energy production (an aerobic cell) must obtain molecular oxygen (O2) from the surrounding medium by diffusion through its plasma membrane. The cell is so small, and the ratio of its surface area to its volume is so large, that every part of its cytoplasm is easily reached by O2 diffusing into the cell. As the size of a cell increases, its surface-to-volume ratio decreases (Fig. 2-2), until metabolism consumes O2 faster than diffusion can supply it. Aerobic metabolism thus becomes impossible as cell size increases beyond a certain point, placing a theoretical upper limit on the size of the aerobic cell.

There are interesting exceptions to this generalization that cells must be small. The giant alga Nitella has cells several centimeters long. To assure the delivery of nutrients, metabolites, and genetic information (RNA) to all of its parts, each cell is vigorously "stirred" by active cytoplasmic streaming (p. 43). The shape of a cell can also help to compensate for its large size. A smooth sphere has the smallest surface-to-volume ratio possible for a given volume. Many large cells, although roughly spherical, have highly convoluted surfaces (Fig. 2-3a), creating larger surface areas for the same volume and thus facilitating the uptake of fuels and nutrients and release of waste products to the surrounding medium. Other large cells (neurons, for example) have large surface-to-volume ratios because they are long and thin, star-shaped, or highly branched (Fig. 2-3b), rather than spherical.

Figure 2-3 Convolutions of the plasma membrane, or long, thin extensions of the cytoplasm, increase the surface-to-volume ratio of cells. (a) Cells of the intestinal mucosa (the inner lining of the small intestine) are covered with microvilli, increasing the area for absorption of nutrients from the intestine. (b) Neurons of the hippocampus of the rat brain are several millimeters long, but the long extensions (axons) are only about 10 nm wide.


Enzymes

16.4.3 Sucrose Synthesis and Degradation

Sucrose is the most abundant disaccharide containing glucose and a fructose molecule as fruit sugars. It is the major form of sugar translocated from photosynthetic to nonphotosynthetic tissues via phloem. Its synthesis occurs in cytoplasm of photosynthetic cells in the following way:

Sucrose is translocated from cytosol of photosynthetic cells to nonphotosynthetic tissues (roots, tubers, and seeds) and may be stored temporarily as sucrose or further converted to starch. For this, sucrose is catalyzed to its monomers by sucrose synthase.

Furthermore, the activated precursor for starch biosynthesis is ADP-glucose, so UDP is replaced with ADP and ADP-glucose can then enter starch synthesis via starch synthase.


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Supplementary Figure 1 Overall structure of drSLC38A9.

a, Central panel, side view in plane of lysosomal membrane. The position of the Fab fragment bound on the luminal side is shown by a gray triangle above the luminal loops. Left panel, cytosolic view showing the vestibule at the cytosolic side. Right panel, luminal view. At the luminal side, residues on TM1b, loop 5–6 and loop 7–8 are grouped in cluster 1 (W128, K131, Q132, R344, E411, P415) and those in TM6a, TM10 and TM11 are grouped in cluster 2 (P348, G491, R495, N542, Q546). An enlarged window from the luminal view encompasses luminal gating cluster 1 and cluster 2. b, The electron density of the determined asymmetric unit of drSLC38A9–Fab crystals. Each asymmetric unit contained two drSLC38A9 molecules (labeled SLC38A9 mol 1 and mol 2) as well as two Fab fragments (labeled Fab mol 1 and Fab mol 2). c,d, Examples of the quality of the electron density maps in the determined structure with fitting of the model of drSLC38A9–Fab.

Supplementary Figure 2 Crystal packing and asymmetric unit of the drSLC38A9–Fab complex.

a, Crystal packing showing the drSLC38A9–Fab complex lattice. Fab fragments (gray) stack tightly along the crystallographic b axis and are connected by drSLC38A9 (cyan) layers in the crystallographic ac plane in a propeller-like head-to-side manner. One asymmetric unit is selected to show the building block that is composed of two Fab (orange) and two drSLC38A9 (red) molecules. b, Interactions between drSLC38A9 and adjacent Fab fragments. One drSLC38A9 (red) makes contacts with four other molecules. The biologically functional contact is between the luminal loops of drSLC38A9 (red) and the complementarity-determining regions (CDRs) of the Fab (orange). The three other contacts, which appear to be crystal contacts and nonspecific, occur between loop 2–3 (red) and TM5 (blue 1) and between TM3, TM10 (red) and a groove shaped from the two adjacent Fab fragments (green 2 and yellow 3).

Supplementary Figure 3 Superposition of drSLC38A9 (colored) and AdiC (3L1L) (translucent yellow).

TM3, TM4, TM8 and TM9 are used in the superimposition. The location of the bound arginine in drSLC38A9 is shown by the dashed oval. Structural alignment was performed in PyMOL. AdiC to drSLC38A9 r.m.s. deviation = 3.57 Å, Cealign for 72 residues.

Supplementary Figure 4 Elution profile of the ΔN-SLC38A9–Fab assembly.

a, The solid line represents the elution trace of the ΔN-SLC38A9–Fab complex and unbound Fab. The dashed line is the elution trace of pure ΔN-SLC38A9. An apparent peak shift was observed for the formation of the ΔN-SLC38A9–Fab complex. b, Fractions selected in the red box in a were sampled and analyzed by SDS–PAGE before being pooled and concentrated for crystallization.



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